Methods, mixtures, kits and compositions pertaining to analyte determination

(Cross-reference with related applications) This application claims the priority of U.S. Provisional Patent Application No. 60 / 443,612 (filed January 30, 2003), which U.S. Patent Application is incorporated herein by reference. (Field of invention) The present invention relates to the field of analysis object measurement by mass spectrometry.

(1. Introduction) The present invention relates to methods, mixtures, kits and / or compositions for measuring one or more analytes by mass spectrometry. The analyte can be any molecule of interest. Non-limiting examples of the analyte include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, lipids, steroids, and small molecules with a molecular weight of less than 1,500 daltons.

Labeling of the analyte can be carried out by reacting the analyte with a labeling reagent that is a compound of formula RP-X-LK-Y-RG or a salt thereof. Here, in the formula, RG is a reactive group that reacts with the analyte, and RP, X, LK, and Y are described in more detail below. Therefore, the labeled analyte can have the general formula: RP-X-LK-Y-analyst. Multiple sets of isomeric or isobaric labeling reagents can be used to label an analyte consisting of two or more different samples. Here, the labeling reagents can be different for each different sample, and the labeling reagents can include a unique reporter (RP) that can bind to the sample that results in the labeled analyte. Thus, information (eg, the presence and / or amount of reporter) can be correlated with the presence and / or amount (often concentration and / or quantity) of the analyte in the sample, which labels different samples. The same can be said for information obtained by analyzing a composite mixture of a plurality of labeled analysts produced by mixing the obtained plurality of products. Analysis of such a complex mixture makes it possible to measure one or more analytes from multiple samples in a single sample or multiple samples. Therefore, the methods, mixtures, kits, and / or compositions of the present invention are particularly well suited for multiplex analysis of composite sample mixtures. For example, they can be used for proteome analysis and / or genomic analysis, as well as correlation studies related to genomic and proteome analysis.

<p> (2. Definition) For the purposes of interpreting this specification, the following definitions apply, and the terms used in the singular form also include the plural form, wherever appropriate, and vice versa. That is, As used herein, "analyte" refers to a molecule of interest that can be measured. Non-limiting examples of analytes include, but are not limited to, proteins, peptides, nucleic acids (both DNA or RNA), carbohydrates, lipids, steroids, and / or small molecules with a molecular weight of less than 1,500 daltons. Can be done. The source of the analysis product or the sample containing the analysis product is not limited, and may be derived from any source. One or more analytes can be natural or synthetic. Non-limiting examples of the analyte or the source of the sample containing the analyte include, but are not limited to, cells or tissues, or cultures (or subcultures) thereof. Non-limiting examples of sources of analysis include, but are not limited to, crude or processed cell lysates, body fluids, tissue extracts, or cell extracts. Yet another non-limiting example of the source of analysis includes, but is not limited to, fractions obtained from separation processes such as chromatographic separation or electrophoretic separation. Body fluids include, but are not limited to, blood, urine, stool, cerebrospinal fluid, cerebrospinal fluid, amniotic fluid, lymph, or glandular secretions. Processed cell lysate means that in addition to the processing required to lyse cells, the processed cell lysate is processed to further process the recovered material. Is Rukoto. For example, a sample is a cell lysate containing one or more analytes that are peptides, the peptide being formed by proteolytic enzyme treatment of all protein components of the crude cell lysate and digestion of precursor proteins. ..</p><p> b. As used herein, "fragmentation" refers to the disruption of covalent bonds.</p><p> c. As used herein, "fragment" means the product of fragmentation (noun) or the product of a (verb) operation that causes fragmentation.</p><p> d. It is well acknowledged that the mass of an atom or molecule can often be close to the closest integer atomic mass unit or the closer 0.1 or 0.01 position of the atomic mass unit. As used herein, "gross mass" refers to the absolute mass as well as the approximate mass within a certain range. Here, a certain range means that very slight differences in mass between the different isotopes used may or may not be detected, but for the purpose of balancing the masses of the reporter and / or linker moieties. Different atomic species so that the isotopes are in the functionally equivalent range (so that the total weight of the reporter / linker combination is the same in the isobaric or isomer labeling reagents of the set or kit). It means that the use of isotopes is similar in terms of mass.</p><p> For example, common isotopes of oxygen have a total mass of 16.0 (actual mass 15.9949) and 18.0 (actual mass 17.9992), and common isotopes of carbon have a total mass of 12.0 (actual mass 12.00000). ) And 13.0 (actual mass 13.00336), and common isotopes of nitrogen are total masses 14.0 (actual mass 14.0031) and 15.0 (actual mass 15.0001). While these values are approximations, those skilled in the art will appreciate one set of reporters.¹⁸When using the O isotope, for example, of the set¹⁶In different reporters, including O, an additional 2 mass units can be supplemented (in addition to oxygen, which has a total mass of 16.0), elsewhere in the reporter.¹²Instead of two C atoms¹³Two C atoms,¹⁴Instead of two N atoms¹⁵Two N atoms, otherwise¹²C and¹⁴Instead of N¹³1 C atom and¹⁵By incorporating one N atom,¹⁸Those skilled in the art understand that O can be supplemented. In this way, the two different reporters in the above set are functional mass equivalents (ie, have the same total mass). Because¹³Two C atoms (¹²(Instead of two C atoms),¹⁵Two N atoms (¹⁴(Instead of two N atoms),¹³With one C atom¹⁵One N (¹²With C atom¹⁴(Instead of N atom), or¹⁸One O atom (¹⁶By using (instead of one O atom), the actual difference in mass that results in a mass increase of 2 Daltons in all of the set or kit labels is a hindrance to the nature of the analysis. Because it does not become.</p><p> This can be illustrated with reference to FIG. In Figure 8, the reporter / linker combination for compound XVII (Figure 8, chemical formula: C).₅¹³CH₁₀¹⁵N₂O) is two¹⁵N atom and one¹³It has a C atom and has a total theoretical mass of 129.138. By comparison, isobaric XV (Fig. 8, formula C)₅¹³CH₁₀N₂¹⁸O) is one¹⁸O atom and one¹³It has a C atom and has a total theoretical mass of 129.151. Compounds XVII and XV are isobars and are structurally and chemically indistinguishable except for their heavy atom isotope content, but with slight differences in absolute mass (mass 129.138 vs. mass 129.151, respectively). However, for the purposes of the present invention, the total mass of compounds XVII and XV is 129.1. Because this total mass hinders the analysis, whether or not the mass spectrometer is sensitive enough to measure the small difference between the absolute mass of the isobaric XVII and the absolute mass of the isobaric XV. Because it does not become.</p><p> From Figure 8, it is clear that the distribution of identical heavy atom isotopes within a single structure is not the only thing to consider in producing a set of isomeric and / or isobaric labeling reagents. is there. It is possible to mix heavy atom isotopes to achieve the desired total weight isomer or isobaric. Thereby, both the selection (combination) of heavy atom isotopes and their distribution are available for study in the production of isomeric and / or isobaric labeling reagents useful for embodiments of the present invention. Is.</p><p> e. As used herein, "isotopically enriched" refers to one or more heavy atom isotopes (eg, deuterium, etc.).¹³C,¹⁵N,¹⁸O,³⁷Cl, or⁸¹A compound (eg, labeling reagent) that is synthetically enriched with a stable isotope such as Br. Since isotopic enrichment is not 100% effective, impurities in compounds with inferior enrichment status are present and the mass is lower. Similarly, due to excess enrichment (unwanted enrichment) and due to the abundance of natural isotopes, higher mass impurities may be present.</p><p> f. As used herein, "labeling reagent" refers to the portion suitable for marking an analyte for measurement. The term label is synonymous with tags and marks, as well as other equivalent terms and phrases. For example, labeled analytes are also referred to as tagged or marked analytes. Therefore, the terms "marker", "tag", "mark", and derivatives of those terms are interchangeable and are suitable parts, or marks, to mark the analyte for measurement. It refers to the attached part.</p><p> g. As used herein, "support," "solid support," or "solid carrier." "carrier)" means any solid phase material on which the labeling reagent can be immobilized. Immobilization is used, for example, to label an analyte or to prepare a labeling reagent, whether or not labeling is done on the support. Solid supports include "resin", "synthetic support", "solid phase", "surface", "membrane", and / or "support". The solid support can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as copolymers and grafts thereof. Similarly, the solid can be inorganic, such as glass, silica, controlled porous glass (CPG), or reverse phase silica. The morphology of the solid support is beads, spheres, particles, granules, gels, membranes, or planar. The surface is planar, substantially planar, or non-planar. The solid support is porous or non-porous and can have swelling or non-swelling properties. The solid support can be a well, recess, or other container, container, appearance or arrangement. Multiple solid supports can be configured in a single sequence for robotic delivery of reagents or at various positions addressable by detection methods and / or instruments.</p><p> h. As used herein, "natural isotope abundance" is one species found in a compound based on the widespread natural prevalence of one or more isotopes in nature. The above isotope level (or distribution). For example, natural compounds obtained from living plants typically contain about 0.6%.¹³Including C.</p><p>(3. Overview) (Reactive group) The reactive group "RG" of the labeling reagent used in the method, mixture, kit, and / or composition embodiment is either an electrophile or a nucleophile and is one or more reactive analysts of a sample. It is possible to react with. Reactive groups can be pre-existing or the reactive groups can be prepared in-situ. In-situ preparation of reactive groups can proceed in the absence of the reactive analyte or in the presence of the reactive analyte. For example, modification of the carboxylic acid group can be done in-situ with a water-soluble carbodiimide (eg, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; EDC), thereby the amino group or the like. Prepare an electrophilic reagent that can react with the nucleophile. In some embodiments, activating the carboxylic acid group of the labeling reagent with EDC can be done in the presence of an analyte containing an amine (nucleophile). In some embodiments, the analyte containing an amine (nucleophile) can also be added after the initial reaction with EDC has taken place. In some embodiments, reactive groups can be produced in-situ by removing the protecting groups in-situ. As a result, any existing or newly produced reagent or plurality of reagents that may result in derivatization of the analyte by the reaction of the nucleophile and / or electrophile are the methods, mixtures, kits, and / of the present invention. Alternatively, it is considered according to the composition embodiment.</p><p> When the reactive group of the labeling reagent is an electrophile, the reactive group can chemically react with a suitable nucleophile in one or more analytes. When the reactive group of the labeling reagent is a nucleophile, the reactive group can react with a suitable electrophilic group of one or more analytes. Numerous pairs of suitable nucleophiles and electroscavenging reagents are known and are often used in the chemical and biochemical technical fields. Appropriate nucleophiles or electrophiles capable of derivatizing them by ligation with reagents (eg, proteins, peptides, nucleic acids, carbohydrates, lipids, steroids, or other small molecules &lt;1,500 daltons). Non-limiting examples of group-containing reagents are available in the literature (Pierce Life Science &amp; Analytical Research Products Catalog &amp; Handbook (a Perstorp Biotec Company), Rockford, IL 61105, USA). Other suitable reagents are well known in the art and are commercially available from many other vendors such as Sigma-Aldrich.</p><p> The reactive group of the labeling reagent can be an amine reactive group. For example, the amine reactive group can be an active ester. An active ester is a specific ester that is well known in peptide synthesis and easily reacts with the amino acid N-α amine under conditions commonly used in peptide synthesis. Amine-reactive active esters include N-hydroxysuccinimidyl ester, N-hydroxysulfosuccinimidyl ester, pentafluorophenyl ester, 2-nitrophenyl ester, 4-nitrophenyl ester, 2,4-dinitrophenyl ester, Alternatively, it can be 2,4-dihalophenyl ester. For example, the alcohol or thiol group of an active ester can have the following formula: That is,</p><p><chemistry num="33"><img file="JP2010190902A_D0001.tif" /></chemistry>In the formula, X is O or S, but preferably O. All of the above are alcohol or thiol groups, which are known to form active esters in the field of peptide chemistry, where the amino acid N-α-amino reacts with the carbonyl carbon of the ester. Is replaced by. It must be revealed that the active ester of any suitable labeling / tagging reagent described herein (eg, N-hydroxysuccinimidyl ester) can be prepared using well-known methods (eg, N-hydroxysuccinimidyl ester). Greg T. Hermanson (1996). See The Chemistry of Reactive Groups in Bioconjugate Techniques Chapter 2 pages 137-165, Academic Press, (New York). Similarly, see Innovation And Perspectives In Solid Phase Synthesis, Editor: Roger Epton, SPCC (UK) Ltd, Birmingham, 1990)). General Formula: A method for forming an active ester of an N-substituted viperazine acetic acid compound, which is a representative example of a labeling reagent for RP-X-LK-Y-RG, is incorporated herein by US Patent Application No. 10 / 751,354 (incorporated herein). )It is described in.</p><p> In another embodiment, the reactive group of the labeling reagent can be a mixed anhydride because the mixed anhydride reacts efficiently with the amino group to form an amide bond.</p><p> The reactive group of the labeling reagent can be a thiol reactive group. For example, the thiol reactive group can be an α-haloacyl halide, an alkyl halide, or a maleimide. Halogenation or halide or halo shall mean an atom of fluorine, chlorine, bromine, or iodine.</p><p> The reactive group of the labeling reagent can be a hydroxyl reactive group. For example, the hydrocyclyl reactive group can be a trityl-halide moiety or a silyl-halide moiety. The trityl-halide reactive moiety can be substituted (eg, Y-methoxytrityl, Y-dimethoxytrityl, Y-trimethoxytrityl, etc.) or unsubstituted, where Y is: Is defined as. The silyl-reactive moiety can be an alkyl-substituted silyl halide, such as Y-dimethylsilyl, Y-ditriethylsilyl, Y-dipropylsilyl, Y-diisopropylsilyl, etc.), where Y is: Defined in.</p><p> The reactive group of the labeling reagent can be a nucleophile such as an amine group, a hydroxy group, or a thiol group.</p><p> (Reporter part) The reporter portion of one labeling reagent or multiple labeling reagents used in methods, mixtures, kits, and / or composition embodiments has a unique measurable mass (or mass-to-charge ratio (mass-to-charge ratio)). )) Is a group. Therefore, each reporter in a set can have a unique total mass. Different reporters can achieve their unique mass by having one or more heavy atom isotopes. For example, carbon isotopes (¹²C,¹³C, and¹⁴C), nitrogen isotope (¹⁴N and¹⁵N), isotope of oxygen (¹⁶O and¹⁸O), or hydrogen isotopes (hydrogen, deuterium, and tritium) are present and can be used to prepare diverse groups of reporter moieties. As an example of a stable heavy atom isotope,¹³C,¹⁵N,¹⁸O, and deuterium can be mentioned. Other light and heavy atom isotopes can also be used in the reporter and are not limited to these. Suitable starting materials for the preparation of reporters containing light and heavy atom isotopes include various sources such as Cambridge Isotope Laboratories, Andover, MA (see the list or "Starting Materials" at www.isotope.com) and It is available from Isotec (a division of Sigma-Aldrich). Cambridge Isotope Laboratories and Isotec will also prepare the desired compounds under a custom synthetic contract. Same as above (Id).</p><p> By associating a unique reporter with a sample of interest, one or more analyzes of that sample can be labeled with the reporter. In this way, the information about the reporter can be associated with the information about one or all of the analyzes of the sample. However, when measuring a reporter, it is not necessary for the reporter to be physically linked to the analyte. Rather, the unique total mass of the reporter can be measured, for example, by fragmenting the ions of the labeled analyte to produce daughter fragment ions and a detectable reporter, followed by a second mass spectrometry of a tandem mass spectrometer. it can. The measured reporter can be used to identify the sample from which the measured analyte is derived. In addition, it is included in one or more samples using a unique amount of reporter when compared to the amount of other reporters or when compared to calibration criteria (eg, an analysis labeled with a particular reporter). Relative or absolute quantities (often expressed as concentrations and / or quantities) of the analyte can be measured. Thus, information (eg, the amount of one or more analytes contained in a particular sample) can be associated with the reporter site used to label each particular sample. When measuring the identity of one or more analytes, the identity of each labeled analyte contained in one or more samples can be correlated by correlating that information with information related to different reporters. And quantity measurement is facilitated.</p><p> The reporter either contains a fixed charge or is capable of being ionized. Since the reporter either contains an immobilized charge or is capable of being ionized, the labeling reagent is isolated or reactive in the form of salts or zwitterions. It can be used to label the analyte. Ionization of the reporter facilitates the measurement of the reporter on a mass spectrometer. Therefore, the reporter is ionized (sometimes signature ion). It can be measured as ion)). When ionized, the reporter can contain one or more net positive or negative charges. Therefore, the reporter can contain one or more acidic or basic groups. This is because such groups can be easily ionized within the mass spectrometer. For example, the reporter may contain one or more basic nitrogen atoms (positively charged) or one or more ionic acidic groups such as carboxylic acid groups, sulfonic acid groups, or phosphoric acid groups (negatively charged). it can. Non-limiting examples of reporters containing basic nitrogen include substituted or unsubstituted morpholiline, piperidine, or piperazine.</p><p> The reporter can be a 5, 6 or 7-membered heterocycle with a ring nitrogen atom N-alkylated by a substituted or unsubstituted acetic acid moiety, via the carbonyl carbon of the N-alkyl acetic acid moiety to this acetic acid moiety. The analytes combine. Here, each different label contains one or more heavy atom isotopes. The heterocycle can be substituted or unsubstituted. The heterocycle can be aliphatic or aromatic. Possible substituents on the heterocyclic moiety include alkyl, alkoxy, and aryl groups. Substituents can include protected or unprotected groups suitable for attaching the analyte to the support, such as amine, hydroxy, or thiol groups. The heterocycle can further comprise one or more heteroatoms such as nitrogen, oxygen, or sulfur atoms.</p><p> Reporter selection can be made under conditions typical of analysis of the analyte, with virtually no sub-fragment. Reporter selection is substantially sub-under the conditions of dissociation energy applied to cause fragmentation at both the bonds X and Y of at least some of the selected ions of the labeled analyte on a mass spectrometer. It can be done so that fragmentation does not occur. "Does not substantially "Sub-fragment)" means that the reporter fragment is difficult or impossible to detect above background noise when applied to a successful analysis of the analysis of interest. The total mass of the reporter is deliberately chosen to be different compared to the mass of the analyte to be measured or compared to the mass of any of the expected fragments of the analyte. For example, if the protein or peptide is an analyte, the total mass of the reporter can be selected differently compared to any naturally occurring amino acid or amino acid, or a predicted fragment thereof. This can facilitate the measurement of the analyte. This is because, depending on the analyte, the lack of any possible component of a sample with a same coincident mass can add confidence to the results of any analysis.</p><p> The reporter can be a small molecule that is not a polymer. The reporter does not have to be a biopolymer (eg, peptide, protein, or nucleic acid) or a component of the biopolymer (eg, amino acid, nucleoside, or nucleotide). The total mass of the reporter is less than 250 daltons. Such small molecules can be easily measured in the second mass spectrometry without the other components of the sample having the same mass in the first mass spectrometry. In this context, a second mass spectrometry can be performed on selected ions measured in the first mass spectrometry, typically with a tandem mass spectrometer. Ions with a particular mass-to-charge ratio can be specifically selected from the first mass spectrometry for possible fragmentation and further mass spectrometry, so that the non-selected ions from the first mass spectrometry are the first. It is not brought into the second mass spectrometry, so there is no contamination in the spectrum of the second mass spectrometry. In addition, the sensitivity of the mass spectrometer and the linearity of the detector (for quantification purposes) can be significantly increased in this low mass range. In addition, the current state of mass spectrometer technology can allow baseline mass resolutions of less than 1 dalton in this mass range (see, eg, Figure 6). These factors may prove to be useful advances for the state of the art.</p><p> (Linker part) The linker moiety of one labeling reagent or multiple labeling reagents used in the method, mixture, kit, and / or composition embodiment may bind the reporter to the analyte, depending on whether a reaction with the analyte has occurred. Alternatively, a reporter is attached to the reactive group. Linker selection should be made to generate a neutral species if both bindings X and Y are fragmented (causing neutral loss for both binding X and Y fragmentation). Can be done. The linker is a very small part, eg a carbonyl or thiocarbonyl group. For example, the linker contains at least one heavy atom isotope and has the following formula. That is,</p><p><chemistry num="34"><img file="JP2010190902A_D0002.tif" /></chemistry>In the formula, R¹Are the same or different and can be an alkyl group containing 1 to 8 carbon atoms, the alkyl group may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein The carbon atoms of the alkyl and aryl groups in contain separately bonded hydrogen, deuterium, and / or fluorine atoms. The linker can also be a larger part. The linker can be a polymer or a biopolymer. The linker can be designed to be sub-fragmented (including sub-fragmentation that produces only the neutral fragment of the linker) when the linker is exposed to dissociation energy levels.</p><p> The linker moiety contains one or more heavy atom isotopes such that their mass compensates for differences in total weight between reporters for each analyte of the mixture, or between reporters for reagent sets and / or kits. be able to.</p><p> In addition, the aggregate gross mass of the reporter / linker combination (ie, the total mass obtained as a whole) is the same for each labeled analyte of the mixture, or for the reagents set and / or kitted. be able to. More specifically, the linker moiety can compensate for differences in total mass between reporters of labeled analytes obtained from different samples. In this case, the intrinsic total mass of the reporter correlates with the sample from which the labeled analyte is derived, and the integrated total mass of the reporter / linker combination is the same for each labeled analyte of the sample mixture regardless of the sample from which it is derived. .. In this way, the total mass of the same analyte in two or more different samples can have the same total mass when producing the sample mixture by labeling and subsequent mixing.</p><p> For example, the reagents in the labeled analyte or set and / or kit for labeling the analyte can be isomers or isobaric. Therefore, if an ion with a particular mass-charge ratio (obtained from the sample mixture) is selected (ie, the selected ion) in a mass spectrometer for initial mass spectrometry of the sample mixture, another component of the sample mixture is therefore selected. The same analyte obtained from the sample is represented by selected ions in proportion to each concentration and / or quantity in the sample mixture. Thus, the linker can not only bind the reporter to the analyte, but can also correct for different masses of the unique reporter moiety, thereby balancing the total mass of the reporter / linker combination in labeled analytes of different masses. ..</p><p> Since the linker can act as a mass balance for the reporter within the labeling reagent, the number of atoms in the linker is such that the integrated total mass of the reporter / linker combination is the same for all reagents in the set or kit. The higher the number, the greater the possible number of different isomer / isobaric labeling reagents in the set and / or kit. In other words, in general, as the number of atoms contained in a linker increases, so does the number of potential reporter / linker combinations that exist. This is because the isotope can be replaced at most anywhere in the linker, which produces an isomer or isobaric of the linker moiety, which offsets the different masses of the reporter moiety. Used in the production of a set of reporter / linker isomers or isotopes. Diverse sets of such multiple labeling reagents are particularly well suited for multiplex analysis of the same and / or different sample analytes.</p><p> The total number of labeling reagents in the set and / or kit is 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The variety of labeling reagents in sets or kits includes the number of atoms in the reporter and linker moieties, the heavy isotopes used to replace the light isotopes, and the various synthetics in which the isotopes are placed synthetically. Limited only by form. However, as already suggested, a number of isotopic-enriched starting materials are readily available from manufacturers such as Cambridge Isotope Laboratories and Isotec. Such isotopic-enriched starting materials can be used to produce sets of isotopic and isomer labeling reagents, or sets of isotopic and isomer labeling reagents. Can be used to produce isotopic-enriched starting materials that can be used in the synthetic processes used to produce. Some examples of the preparation of isobaric labeling reagents suitable for use with a set of labeling reagents can be found in the Examples section below.</p><p> (Reporter / linker combination) The labeling reagents described herein are composed of a reporter and a linker, which are bound via bond X. As mentioned above, the reporter / linker combination has the same total mass for each component of the labeling reagents in one set and / or kit. In addition, the binding X of the labeling reagent reporter / linker combination is designed to fragment at least a portion of the selected ions upon subject to dissociation energy levels, thereby releasing the reporter from the analyte. can do. Therefore, the total mass of the reporter (as an m / s ratio) and its intensity can be observed by MS / MS analysis.</p><p> Reporter / linker combinations can include different combinations of the same or different heavy atom isotopes between the various labeling reagents in the set or kit. In the scientific literature, this was sometimes referred to as coding or isotope coding. For example, Abersold et al. Disclosed isotope coded affinity tags (see ICAT; WO 00/11208). In some respects, Abersold et al. Are not the labeling reagents of the invention because Abersold does not teach two or more equal mass labeling reagents such as isomeric or isobaric labeling reagents. different.</p><p> (Mass Spectrometer / Mass Spectrometry (MS)) The method of the present invention can be carried out using a tandem mass spectrometer and another mass spectrometer capable of selecting and fragmenting molecular ions. A tandem mass spectrometer (and a lesser degree single-stage mass spectrometer) results in the selection and fragmentation of molecular ions based on the mass-to-charge (m / z) ratio. Has the ability to record fragment (daughter) ion spectra. More specifically, a daughter fragment ion spectrum can be generated by subjecting the selected ion to a dissociation energy level (eg, collision-induced dissociation (CID)). For example, the ions corresponding to the labeled peptide of a particular m / z ratio can be selected from the first mass spectrometry, fragmented and further analyzed again in the second mass spectrometry. Typical devices capable of performing such tandem mass spectrometry are, but are not limited to, magnetic field 4-sector, tandem time-of-flight, triple quadrupole, ion trap, and hybrid quadrupole flight time. (Q-TOF) Mass spectrometer can be mentioned.</p><p> These types of mass spectrometers may be used with various ion sources. Such sources of ionization include, but are not limited to, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The ionization source can be used to generate a charge species for a first mass spectrometry where the analyte does not have a fixed charge in advance. Additional mass spectrometric instruments and fragmentation methods include post-source decay in MALDI-MS instruments and high-energy CIDs using MALDI-TOF (time-of-flight) -TOF MS. For a recent overview of tandem mass spectrometers, see R. Aebersold and D. Goodlett, Mass Spectrometry in Proteomics. Chem. Rev. 101: 269-295 (2001). See also U.S. Pat. No. 6,319,476 (incorporated herein) for a discussion of TOF-TOF mass spectrometry techniques.</p><p> (Fragmentation by dissociation energy level) It is well understood that binding can be fragmented as a result of the process that occurs in a mass spectrometer. In addition, bond fragmentation is the result obtained on a mass spectrometer by exposing the ions to dissociation energy levels. For example, dissociation energy levels can be generated within a mass spectrometer by collision-induced dissociation (CID). Those skilled in the art of mass spectrometry understand that other typical techniques for improving the dissociation energy that causes fragmentation include, but are not limited to, photodissociation, electron capture, and surface-induced dissociation. To do.</p><p> The process of fragmenting the bond by collision-induced dissociation also involves enhancing the kinetic energy state of the selected ion to the point where bond fragmentation occurs through collision with an inert gas. For example, kinetic energy can be transferred by collision with an inert gas (eg, nitrogen, helium, or argon) within the collision cell. The amount of kinetic energy that can be transferred to an ion is proportional to the number of gas molecules allowed to enter the collision cell. If more gas molecules are present, more kinetic energy is transferred to the selected ions, and if fewer gas molecules are present, lower kinetic energy is transferred.</p><p> Therefore, it is clear that the dissociation energy level in the mass spectrometer can be controlled. It is also well recognized that some bonds are more unstable than others. Binding instability at the analyte or reporter / linker moiety depends on the nature of the analyte or reporter / linker moiety. Thus, dissociation energy levels can be controlled so that the analyte and / or label (eg, reporter / linker combination) can be fragmented in a measurable manner. How to make such routine adjustments to the components of the mass spectrometer will be understood by those of skill in the art, thereby achieving the appropriate level of dissociation energy and of the ions of the labeled analyte. At least a portion is fragmented into an ionization reporter portion and daughter fragment ions.</p><p> For example, the dissociation energy can be applied to the ions selected / separated from the first mass spectrometry. In a tandem mass spectrometer, the extracted ions are subjected to dissociation energy levels and transferred to a second mass spectrometer. The selected ions can have a selected mass-to-charge ratio. The mass-to-charge ratio can be within the range of the mass-to-charge ratio depending on the characteristics of the mass spectrometer. When collision dissociation is used, the ions can be transferred from a first mass spectrometer to a second mass spectrometer by passing the ions through a collision cell to which dissociation energy can be applied, thereby fragmenting. Ions are generated. For example, the ions sent to the second mass spectrometer for analysis may include some or some residual (non-fragmented) selected ions, the daughter fragment ions of the labeled analyte and the reporter. -Can be included in the same way as ions (sign ions).</p><p> (Analyst measurement by computer-assisted database analysis) In some embodiments, the analyte can be measured based on a daughter ion fragmentation pattern. Here, fragmentation / patterns are analyzed by computer-assisted comparison with the spectrum of known or "theoretical" analytes. For example, the daughter fragment ion spectrum of a peptide ion fragmented under low energy CID conditions is considered the sum of many discrete fragmentation events. A common nomenclature distinguishes daughter fragment ions based on the degrading amide bond and the peptide fragment that retains the charge after cell division. Charge retention on the N-terminal side of the fissionable amide bond results in the formation of b-type ions. If a charge remains on the C-terminal side of the broken amide bond, the fragment ion is called a y-type ion. In addition to the b and y type ions, the CID mass spectrum may include other diagnostic fragment ions (daughter fragment ions). These include ions produced by the neutral loss of ammonia (-17amu) from glutamine, lysine, and arginine or the loss of water (-18aum) from hydroxyl-containing amino acids such as serine and threonine. It has been observed that some amino acids are more easily fragmented under conditions of lower energy CID than others. This is especially true for peptides containing proline or aspartic acid residues, and even more so for aspartyl-proline bonds (Mak, M. et al., Rapid Commun. Mass Spectrom., 12 :). 837-842) (1998). Thus, a Z-pro dimer or Z-asp dimer peptide bond (where Z is any natural amino acid, pro is proline, and asp is aspartic acid) is a peptide bond between all other amino acid dimer combinations. Compared to, it tends to be more unstable.</p><p> Thus, for peptide and protein samples, the low-energy CID spectrum contains redundant sequence-specific information for overlapping b- and Y-based ions, internal fragment ions from the same peptide, and immonium and other neutral loss ions. .. Interpreting such a CID spectrum to construct a new amino acid sequence for the parent peptide is challenging and time consuming. The most striking advance in identifying peptide sequences has been the development of computer algorithms that correct the peptide CID spectrum with peptide sequences already present in protein and DNA sequence databases. Such approaches include SEQUEST (Eng, J. et al. J. Anx. Soc. Mass Spectrom., 5: 976-989 (1994)) and MASCOT (Perkins, D. et al. Electrophoresis, 20: 3551-). Illustrated by programs such as 3567 (1999)).</p><p> In short, the experimental peptide CID spectrum (MS / MS spectrum) matches or correlates with the "theoretical" daughter fragment ion spectrum calculated from peptide sequences obtained from protein or genomic sequence databases. The match or correlation is based on the similarity between the predicted and observed masses of daughter fragment ions in MS / MS mode. Potential matches or correlations are recorded based on how well the experimental and "theoretical" fragment patterns match. Database constraints for searching for a given peptide / amino acid sequence are so distinct that the single peptide CID spectrum is suitable for identifying any given protein in the entire genome or expressed sequence tag (EST) database. For another overview, see Yates, JR Trends, Genetics, 16: 5-8 (2000) and Yates, JR, Electrophoresis 19: 893-900 (1998).</p><p> Therefore, daughter fragment ion analysis of the MS / MS spectrum is used not only to measure the analyte of the labeled analyte, but also to measure the analyte from which the measured analyte is derived. For example, daughter fragment ion analysis of MS / MS spectra can be used to measure proteins in which peptides are cleaved as a result of enzymatic digestion of the protein. It is conceivable that such analysis may be applicable to other analytes, such as nucleic acids.</p><p> (Join X and Y) X is the bond between the atom of the reporter and the atom of the linker. Y is the bond between the linker atom and any of the reactants if the reactive group or labeling reagent is reacting with the reactant. The binding X and Y of the various labeling reagents (ie, RP-X-LK-Y-RG) that can be used in embodiments of the invention are at least the selected ions when exposed to dissociation energy levels. In part, it can be fragmented. Therefore, the dissociation energy level is adjusted with a mass spectrometer so that the bonds X and Y are fragmented into at least a portion of the selected ions of the labeled analyte (ie, the RP-X-LK-Y-analyst). can do. Fragmentation of binding X separates the reporter from the analysis so that the reporter can be measured separately from the analysis. Fragmentation of bond Y separates the reporter / linker combination from the analyzer or the linker from the analyzer, depending on whether bond X is already fragmented. Bond Y can be more destabilized than bond X. Bond X can be more destabilized than Bond Y. Both bonds X and Y can also be relatively unstable.</p><p> If the analyte of interest is a protein or peptide, the relative instability of the bonds X and Y can be regulated with respect to the amide (peptide) bond. Bond X, bond Y, or both bond X and bond Y can be destabilized more, equal, or less compared to typical amide (peptide) bonds. For example, under the condition of dissociation energy, bond X and / or bond Y is a z-pro dimer or z-asp dimer (where Z is any natural amino acid, pro is proline, and asp is aspartic acid). Fragmentation tends to be inferior compared to peptide bonds in. In some embodiments, the bonds X and Y have approximately equal dissociation energies as compared to typical amide bonds. In some embodiments, the bonds X and Y have even higher levels of dissociation energy compared to typical amide bonds.</p><p> Bonds X and Y can also exist such that the fragmentation of bond Y induces fragmentation of bond X and vice versa. In this way, both bindings X and Y can be fragmented substantially simultaneously, with a significant amount of analyte or its daughter fragment ions free of partial labeling in the second mass spectrometry. it can. By "substantial amount of analyte" is meant that less than 25%, preferably less than 10%, partially labeled analyte is measured in the MS / MS spectrum.</p><p> This feature allows from computer-assisted analysis of the daughter fragment ion spectrum, as a clear division between labeled and unlabeled fragments of the analyte lies in the spectrum of the second mass spectrometry (MS / MS). The identification of the analyte can be simplified. In addition, in some embodiments, fragment ions of the analyte can be fully or unlabeled (but not partially labeled) by the reporter / linker moiety, resulting from isotopic distribution across split bonds. The mass of the daughter fragment ions is little or not scattered, for example, on each side of the unstable single bond of the partially labeled assay, which is usually determined by second mass spectrometry. It is conceivable that isotopes are present.</p><p> (Sample processing) In some embodiments of the invention, the sample can be processed prior to labeling as well as after labeling of the analyte. This processing can facilitate labeling of the analyte. This processing can facilitate the analysis of sample components. This processing can simplify the handling of the sample. This process can facilitate two or more of the above.</p><p> For example, the sample can be treated with an enzyme. Enzymes are proteases (to degrade proteins and peptides), nucleases (to degrade nucleic acids), or some other enzyme. Enzyme selection can be made to have a fairly predictable degradation pattern. Two or more proteases and / or two or more nuclease enzymes may be used together, or other enzymes may be used together, which results in degradation of the sample components.</p><p> For example, the proteolytic enzyme trypsin cleaves the peptide bond between lysine or arginine and a non-specific amino acid to cleave the peptide bond between the amine terminal (N-terminal) and the lysine or arginine carboxyl-terminal amino acid (C-terminal). It is a serine protease that produces a peptide having. In this way, the peptide produced by cleavage of the protein is predictable, and its presence and / or quantity can indicate the presence and / or quantity of the protein from which the sample is derived from the trypsin-digested sample. In addition, the free amine terminal of the peptide can be a good nucleophile to promote labeling. Other typical proteolytic enzymes include papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin, and carboxypeptidase C.</p><p> For example, a protein (eg, protein Z) can produce three types of peptides (eg, peptides B, C, and D) when digested with a protease such as trypsin. Therefore, it can be said that a sample that has been digested by a proteolytic enzyme such as trypsin and that has been confirmed to contain peptides B, C, and D by analysis originally constitutes protein Z. The amount of peptides B, C, and D also correlates with the amount of protein Z contained in the digested sample. In this way, any measurement that identifies and / or quantifies one or more of the peptides B, C, and D contained in a sample (or its fraction) is in the original sample (or its fraction). It can be used to identify and / or quantify the protein Z contained.</p><p> Since it is possible to predict the activity of an enzyme, it is possible to predict the sequence of a peptide produced by degrading a protein whose sequence is known. This information produces "theoretical" peptide information. Therefore, the measurement of "theoretical" peptide fragments by computer-assisted analysis of daughter fragment ions (as described above) obtained from mass spectrometry of actual samples is performed by one type contained in one or more unknown samples. It can be used for the measurement of the above peptides or proteins.</p><p> (Separation of sample mixture) In some embodiments, processing of the sample or sample mixture of labeled analyte can include separation. For example, one sample mixture can be prepared that contains a plurality of differentially labeled analyzes from a plurality of different samples. Differentially labeled means that each of the labels has unique identifiable properties (eg, including a unique reporter portion that creates a unique "sign ion" in MS / MS spectrometry). .. To analyze the sample mixture, the components of the sample mixture can be separated and mass spectrometry can be performed on only one fraction of the sample mixture. In this way, the mass of the separated analytes can be analyzed individually to increase the sensitivity of the analytical process, which can substantially reduce the complexity of the analysis. Of course, this analysis can be repeated one or more times for one or more different fractions of the sample mixture to analyze all fractions of the sample mixture.</p><p> Samples with the same multiple differentially labeled analytes co-eluting at a concentration or amount proportional to their abundance in the sample mixture, provided that the amount of each sample added to the sample mixture is known. It can be used to measure the amount of each labeled analyte in each sample containing the mixture. Thus, in some embodiments, separation of the sample mixture can simplify the analysis, while the sample mixture was measured by mass spectrometry with multiple differentially labeled analysts (eg, MS / MS analysis). The correlation between the signals can be maintained.</p><p> The above separation can be performed by chromatography. For example, liquid chromatography / mass spectrometry (LC / MS) can be used to perform separation and mass spectrometry of such samples. In addition, any chromatographic separation process suitable for the separation of the analyte of interest can be used. For example, the chromatographic separation can be normal phase chromatography, reverse phase chromatography, ion exchange chromatography, size exclusion chromatography, or affinity chromatography.</p><p> The above separation can be performed electrophoretically. Non-limiting examples of electrophoretic separation techniques that can be used include, but are not limited to, separation by one-dimensional electrophoresis, separation by two-dimensional electrophoresis, and / or separation by capillary electrophoresis.</p><p> Multiple analyzes of a sample can be labeled with one isobaric labeling reagent or a set of reagents. Multiple isobaric labeling reagents are useful when performing separation steps. This is because the isobaric labeling of a set of labeling reagents is structurally and chemically distinguishable (and by total mass until fragmentation removes the reporter from the analyte). .. Thus, all analytes of the same composition labeled with different isobaric labels can be chromatographed in exactly the same way (ie, co-eluting). Because they are structurally and chemically distinguishable, the eluate produced by the separation process is a fixed amount of each isobaric labeled analyte that is proportional to the amount of labeled analyte contained in the sample mixture. Can be included. In addition, from the knowledge of how the sample mixture was prepared (sample proportions, any other components added to obtain the sample mixture (eg, calibration criteria)), labeling analysis contained in the sample mixture. It is possible to match the amount of material to the original amount of the labeled analyte contained in the sample from which it is derived.</p><p> The labeling reagent can also be isomerized. Although it is sometimes possible to separate the isotopes by chromatography, there are conditions that are condition-dependent, in which the separation process is performed to co-elut all of the multiple identically labeled analyzes. Here, the amounts of all labeled analytes are present in proportion to their concentration and / or quantity in the sample mixture.</p><p> As used herein, isobaric is different from isotope. Isobars cannot be structurally and chemically distinguished from compounds with the same nominal total mass (see, eg, Figure 1) (excluding isotope content and / or distribution). In contrast, isomers can structurally and / or chemically distinguish between isomers even though they have the same nominal total mass.</p><p> (Relative and absolute quantification of the analyte) In some embodiments, one sample mixture allows relative quantification of the same differentially labeled analyte. Relative quantification of the same differentially labeled analyzer is the relative amount of reporter measured in the second mass spectrometry to the selective labeling assay observed in the first mass spectrometry (eg, the area of the reported peak). Or height) is possible by comparison. In other words, if the information for a particular sample used to generate the sample mixture can be correlated with each reporter, then the relative amount of that reporter with respect to the other reporters observed in the second mass spectrometry. Is the relative amount of the analyte contained in the sample mixture. If the components combined to form the sample mixture are known, the relative amount of analyte contained in each sample used to prepare the sample mixture was selected from the first mass spectrometry. It can be calculated based on the original relative amount of the reporter observed for the ions of the labeled analyte. This process can be repeated for all of the different labeled analytes observed in the first mass spectrometry. In this way, the relative amount (often expressed in concentration and / or quantity) of each reactive analyte contained in each of a number of different samples used to generate the sample mixture can be measured. it can.</p><p> In other embodiments, the absolute quantification of the analyte can be measured. For these embodiments, a known amount of one or more differentially labeled analytes (one calibration criterion or multiple calibration criteria) can be added to the sample mixture. The calibration criteria are the expected analytes, which are unique to the reporter for the calibration criteria compared to any of the multiple samples used to form the sample mixture. Labeled with an isomer or homomeric label of the labeling set used to label the analyte of the sample mixture, provided that there is. Once the relative amount of the reporter for the calibration reference is measured with respect to the relative amount of the reporter of the differentially labeled analyte of the sample mixture, all absolute quantities of the differentially labeled analyte contained in the sample mixture (often). It is possible to calculate (expressed in concentration and / or quantity). In this way, the absolute amount of each differential labeled analyte (the sample from which the analyte is derived has a calibration criterion) is also measured based on the knowledge of how the sample mixture was prepared.</p><p> Despite the above, if necessary, the isotope content in naturally occurring or artificially created reporters can be corrected for the intensity of the reporter (sign ion). An example of such a correction can be found in Example 3. A sophisticated example of such correction is also a co-pending and shared United States entitled "Method and MFP For De-Convoluting A Convoluted Spectrum". It can be found in Provisional Patent Application No. 60 / 524,844 (filed November 26, 2003). Greater care is taken to accurately quantify the intensity of each reporter, resulting in more accurate quantification of the relative and absolute quantities of the analyte in the original sample.</p><p> (Proteomix analysis) The methods, mixtures, kits, and / or compositions of the present invention can be used for complex analysis. This is because mass spectrometry techniques can be used to quickly and repeatedly multiplex, analyze, and reanalyze samples. For example, analysis of a sample mixture can be performed on the amount of individual analyte contained in one or more samples. The amount of these analytes (often expressed in concentration and / or quantity) can be measured for the samples that make up the sample mixture. Since sample processing and mass spectrometry can be performed quickly, by repeating these methods many times, the amount of many differentially labeled analytes in the sample mixture can be reduced to their relative amounts in the sample from which the analyte is derived. / Or can be measured in terms of absolute quantity.</p><p> One application where such rapid multiplex analysis is useful is in the area of proteomix analysis. Proteomics can be seen as an experimental approach to explain the information encoded in genomic sequences regarding the regulation of structure, function, and biological processes. This can be achieved by systematic analysis of the total protein components expressed by cells or tissues. Mass spectrometry, used in combination with the methods, mixtures, kits, and / or composition embodiments of the invention, is one of the possible tools for such overall protein analysis.</p><p> For example, with a set of four isobaric labeling reagents, to measure upregulation and downregulation of proteins in a single experiment, eg, based on the response of proliferating cells to a particular stimulant, 4 It is possible to get a point in time. It is possible to incorporate one or two controls, although it can be performed at a smaller time point. In all cases, up or down regulation of protein expression can be measured in a single multiplexing experiment, optionally with respect to the control group. In addition, the results are directly comparable as the machining is performed in parallel. This is because there is no risk that small changes in the protocol could affect the outcome.<u style="single">(Item 1)</u><u style="single">the following:</u><u style="single"> a) A step of reacting two or more samples, each containing one or more reactants, with a different set of labeling reagents, each of which is one or more. Two or more differentially labeled samples containing the labeled analytes of the above were generated, and each of the above different labeling reagents in the above set had the following formula:</u><u style="single"> RP-X-LK-Y-RG</u><u style="single">Or its salt (in the formula,</u><u style="single"> i) RG is a reactive group that is a nucleophile or an electrophile, and can react with one or more of the above-mentioned reactive analysts of the above-mentioned sample.</u><u style="single"> ii) RP includes or a fixed charge or repo capable of ionizing a Ta portion, wherein the mass of each reporter is assumed different for each reagent of the set,</u><u style="single"> iii) LK is a linker moiety that binds the reaction group and the reporter group, and the mass of the linker is the integrated total mass of the combination of the reporter and the linker with respect to each reagent in the set. Correct the difference in total mass between the reporters for the different labeling reagents in the above set so that they are the same.</u><u style="single"> iv) X is the bond between the reporter atom and the linker,</u><u style="single"> v) Y is the bond between the atom of the linker and the atom of the reactive group, where when the labeling reagent reacts with the reactive analyte, the bond Y attaches the linker to the analyte. Combine,</u><u style="single"> vi) Bond X and Bond Y are fragmented at least in at least a portion of the labeled analyte) upon receiving a dissociation energy level);</u><u style="single"> b) A step of mixing the above two or more types of differentially labeled samples or parts thereof with optionally one or more types of calibration criteria to produce a sample mixture, wherein the RP is:</u><u style="single"> i) Have a total mass of less than 250 daltons and / or</u><u style="single"> ii) Substantially no sub-fragmentation and / or under the conditions of dissociation energy applied to cause at least some fragmentation of the bond X and Y of the labeled analyte on a mass spectrometer. ,</u><u style="single"> iii) A process that is not a polymer or a biopolymer,</u><u style="single">Including, methods.</u><u style="single">(Item 2)</u><u style="single">the following:</u><u style="single"> a) A step of reacting two or more samples, each containing one or more reactants, with a different set of labeling reagents, each of which is one or more. Two or more differentially labeled samples containing the labeled analytes of the above were generated, and each of the above different labeling reagents in the above set had the following formula:</u><u style="single"> RP-X-LK-Y-RG or its salt (in the formula,</u><u style="single"> i) RG is a reactive group that is a nucleophile or an electrophile, and can react with one or more of the above-mentioned reactive analysts of the above-mentioned sample.</u><u style="single"> ii) The RP is a reporter moiety that contains a fixed charge or is capable of being ionized, where the mass of each reporter shall be different for each reagent in the above set.</u><u style="single"> iii) LK is a linker moiety that binds the reaction group and the reporter group, and the mass of the linker is the integrated total mass of the combination of the reporter and the linker with respect to each reagent in the set. Correct the difference in total mass between the reporters for the different labeling reagents in the above set so that they are the same.</u><u style="single"> iv) X is the bond between the reporter atom and the linker,</u><u style="single"> v) Y is the bond between the atom of the linker and the atom of the reactive group, where when the labeling reagent reacts with the reactive analyte, the bond Y attaches the linker to the analyte. Combine,</u><u style="single"> vi) Bond X and Bond Y are fragmented at least in at least a portion of the labeled analyte) upon receiving a dissociation energy level);</u><u style="single"> b) A step of mixing the above two or more types of differentially labeled samples or parts thereof and optionally one or more types of calibration criteria to produce a sample mixture, wherein the linker LK Is a process that undergoes neutral loss under the conditions of applied dissociation energy, which causes bond X and Y fragmentation on a mass spectrometer.</u><u style="single">Including, methods.</u><u style="single">(Item 3)</u><u style="single">the following:</u><u style="single"> a) A step of reacting two or more samples, each containing one or more reactants, with a different set of labeling reagents, each of which is one or more. Two or more differentially labeled samples containing the labeled analytes of the above were generated, and each of the above different labeling reagents in the above set had the following formula:</u><u style="single"> RP-X-LK-Y-RG or its salt (in the formula,</u><u style="single"> i) RG is a reactive group that is a nucleophile or an electrophile, and can react with one or more of the above-mentioned reactive analysts of the above-mentioned sample.</u><u style="single"> ii) The RP is a reporter moiety that contains a fixed charge or is capable of being ionized, where the mass of each reporter shall be different for each reagent in the above set.</u><u style="single"> iii) LK is a linker moiety that binds the reaction group and the reporter group, and the mass of the linker is the integrated total mass of the combination of the reporter and the linker with respect to each reagent in the set. Correct the difference in total mass between the reporters for the different labeling reagents in the above set so that they are the same.</u><u style="single"> iv) X is the bond between the reporter atom and the linker,</u><u style="single"> v) Y is the bond between the atom of the linker and the atom of the reactive group, where when the labeling reagent reacts with the reactive analyte, the bond Y attaches the linker to the analyte. Combine,</u><u style="single"> vi) Bond X and Bond Y are fragmented at least in at least a portion of the labeled analyte) upon receiving a dissociation energy level);</u><u style="single"> b) A step of producing a sample mixture by mixing the above two or more types of differentially labeled samples or parts thereof with optionally one or more types of calibration criteria.</u><u style="single">Including,</u><u style="single"> A method, characterized in that one fragmentation of bond X or Y induces fragmentation of the other bond X or Y under the conditions of dissociation energy applied on a mass spectrometer.</u><u style="single">(Item 4)</u><u style="single">the following:</u><u style="single"> a) A step of reacting two or more samples, each containing one or more reactants, with a different set of labeling reagents, each of which is one or more. Two or more differentially labeled samples containing the labeled analyte of the above were generated, where each of the above different labeling reagents in the above set had the following formula: RP-X-LK-Y-RG</u><u style="single"> Or its salt (in the formula,</u><u style="single"> i) RG is a reactive group that is a nucleophile or an electrophile, and can react with one or more of the above-mentioned reactive analysts of the above-mentioned sample.</u><u style="single"> ii) The RP is a reporter moiety that contains a fixed charge or is capable of being ionized, where the mass of each reporter shall be different for each reagent in the above set.</u><u style="single"> iii) LK is a linker moiety that binds the reaction group and the reporter group, and the mass of the linker is the integrated total mass of the combination of the reporter and the linker with respect to each reagent in the set. Correct the difference in total mass between the reporters for the different labeling reagents in the above set so that they are the same.</u><u style="single"> iv) X is the bond between the reporter atom and the linker,</u><u style="single"> v) Y is the bond between the atom of the linker and the atom of the reactive group, where when the labeling reagent reacts with the reactive analyte, the bond Y attaches the linker to the analyte. Combine,</u><u style="single"> vi) Bond X and Bond Y are fragmented at least in at least a portion of the labeled analyte) upon receiving a dissociation energy level);</u><u style="single"> b) A step of mixing the above two or more kinds of differentially labeled samples or a portion thereof with one or more kinds of calibration criteria optionally, and a step of producing a sample mixture by the step.</u><u style="single">Including,</u><u style="single"> i) Under the conditions of the dissociation energy applied by the mass spectrometer, bond X is less prone to fragmentation than bond Y and / or</u><u style="single"> ii) Under the conditions of the dissociation energy applied by the mass spectrometer, the bound X is less prone to fragmentation than the peptide bond of the Z-pro amino acid dimer or the Z-asp amino acid dimer, where Z is A method, characterized in that any natural amino acid, pro is proline and asp is aspartic acid.</u><u style="single">(Item 5)</u><u style="single">the following:</u><u style="single"> a) A step of reacting two or more samples, each containing one or more reactants, with a different set of labeling reagents, each of which is one or more. Two or more differentially labeled samples containing the labeled analytes of the above were generated, and each of the above different labeling reagents in the above set had the following formula:</u><u style="single"> RP-X-LK-Y-RG or its salt,</u><u style="single">(During the ceremony,</u><u style="single"> i) RG is a reactive group that is a nucleophile or an electrophile, and can react with one or more of the above-mentioned reactive analysts of the above-mentioned sample.</u><u style="single"> ii) The RP is a reporter moiety that contains a fixed charge or is capable of being ionized, where the mass of each reporter shall be different for each reagent in the above set.</u><u style="single"> iii) LK is a linker moiety that binds the reaction group and the reporter group, and here,</u><u style="single">a) The mass of the linker is the total mass between the reporters for the different labeling reagents in the set so that the integrated total mass of the combination of the reporter and the linker is the same for each reagent in the set. Correct the difference and</u><u style="single">b) The above linker contains at least one heavy atom isotope and has the following formula:</u><chemistry num="1"><img file="JP2010190902A_D0003.tif" /></chemistry><u style="single">Have,</u><u style="single"> In the formula, R</u>^{<u style="single">1</u>}<u style="single">Is the same or different, and is an alkyl group containing 1 to 8 carbon atoms, and the alkyl group may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the alkyl group is described herein. And the carbon atoms of the aryl group separately contain bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single"> iv) X is the bond between the reporter atom and the linker,</u><u style="single"> v) Y is the bond between the atom of the linker and the atom of the reactive group, where when the labeling reagent reacts with the reactive analyte, the bond Y attaches the linker to the analyte. Including), steps;</u><u style="single"> b) A step of mixing the above two or more kinds of differentially labeled samples or a portion thereof with one or more kinds of calibration criteria optionally, and a step of producing a sample mixture by the step.</u><u style="single">Including, methods.</u><u style="single">(Item 6)</u><u style="single">the following:</u><u style="single"> c) The step of performing the first mass spectrometry on the sample mixture or its fraction;</u><u style="single"> d) Ionized reporter moieties and ionized daughter fragment ions of at least a plurality of the selected ions by exposing the ions of the labeled analyte selected in the first mass spectrometry to dissociation energy levels. And the process of forming;</u><u style="single"> e) The step of performing a second mass spectrometry on the selected ions, the ionized reporter moieties, and the daughter fragment ions, or fractions thereof.</u><u style="single"> The method according to any one of items 1 to 5, further comprising.</u><u style="single">(Item 7)</u><u style="single">the following:</u><u style="single"> f) In the second mass spectrometry, the step of measuring the total mass and relative mass of each reporter portion and the total mass of the daughter fragment ions.</u><u style="single"> The method according to item 6, further comprising.</u><u style="single">(Item 8)</u><u style="single">7. The method of item 7, further comprising repeating steps (d) to (f) one or more times on the ions of the labeled analyte selected with different selected masses to charge ratios.</u><u style="single">(Item 9)</u><u style="single">8. The method of item 8, further comprising repeating steps (a) to (f) one or more times, each time using a different fraction of the sample mixture.</u><u style="single">(Item 10)</u><u style="single">The method according to any one of items 1 to 5, wherein the two or more kinds of samples are products of an enzymatic digestion reaction.</u><u style="single">(Item 11)</u><u style="single">The method according to item 10, wherein the two or more types of samples are products of a proteolytic enzymatic digestion reaction.</u><u style="single">(Item 12)</u><u style="single">The method of item 11, wherein the protein enzyme is trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin, or carboxypeptidase C.</u><u style="single">(Item 13)</u><u style="single">The method according to any one of items 1 to 5, wherein each sample is a crude or processed cell lysate, body fluid, tissue extract, or cell extract.</u><u style="single">(Item 14)</u><u style="single">The method according to any one of items 1 to 5, wherein each sample is a fraction obtained from the separation process.</u><u style="single">(Item 15)</u><u style="single">The method of item 14, wherein the separation process is chromatographic or electrophoretic separation.</u><u style="single">(Item 16)</u><u style="single">13. The method of item 13, wherein the body fluid is blood, urine, cerebrospinal fluid, cerebrospinal fluid, amniotic fluid, lymph, or glandular secretions.</u><u style="single">(Item 17)</u><u style="single">The method according to any one of items 1 to 5, wherein the one or more analytes are a protein, a nucleic acid molecule, a hydrocarbon, a lipid, a steroid, or a small molecule of less than 1500 daltons.</u><u style="single">(Item 18)</u><u style="single">The method according to any one of items 1 to 5, wherein the one or more kinds of the above-mentioned analytes are peptides.</u><u style="single">(Item 19)</u><u style="single">The method of item 18, wherein the peptide is formed by digestion of at least one protein.</u><u style="single">(Item 20)</u><u style="single">19. The method of item 19, wherein the peptide is formed by digestion of all protein components of crude cell lysates.</u><u style="single">(Item 21)</u><u style="single">The method of any one of items 1-5, wherein the reactive groups of each reagent in the set are prepared in situ for reaction with the reactive analyte.</u><u style="single">(Item 22)</u><u style="single">The method of item 21, wherein the reactive group of each of the reagents in the set is a carboxylic acid group activated with a water-soluble carbodiimide.</u><u style="single">(Item 23)</u><u style="single">The method of item 22, wherein the water-soluble carbodiimide is 1- (3-dimethylaminopropyl) -3 ethylcarbodiimide hydrochloride (EDC).</u><u style="single">(Item 24)</u><u style="single">The method according to any one of items 1 to 5, wherein the reactive group of each reagent in the set is an amine-reactive active ester group.</u><u style="single">(Item 25)</u><u style="single">The active ester is N-hydroxysuccinimidyl ester, N-hydroxysulfosuccinimidyl ester, pentafluorophenyl ester, 2-nitrophenyl ester, 4-nitrophenyl ester, 2,4-dinitrophenyl ester, or 2,4. -The method of item 24, which is a dihalophenyl ester.</u><u style="single">(Item 26)</u><u style="single">The method according to any one of items 1 to 4, wherein the reactive group of each reagent in the set is a thiol reaction electrophilic group.</u><u style="single">(Item 27)</u><u style="single">26. The method of item 26, wherein the thiol reactive group is selected from the group consisting of maleimides, alkyl halides, aryl halides, and α-haloacyls.</u><u style="single">(Item 28)</u><u style="single">The method according to any one of items 1 to 4, wherein the reactive group of each reagent in the set is a hydroxyl reaction electrophilic group.</u><u style="single">(Item 29)</u><u style="single">The method according to any one of items 1 to 4, wherein the reactive group of each reagent is a nucleophile selected from the group consisting of an amine group, a hydroxyl group, or a thiol group.</u><u style="single">(Item 30)</u><u style="single">The method according to any one of items 1 to 5, wherein the reporter is a substituted or unsubstituted morpholine, piperidine, or piperazine compound, or a salt thereof.</u><u style="single">(Item 31)</u><u style="single">The method according to any one of items 1 to 5, wherein the reporter is a carboxylic acid, a sulfonic acid, a phosphoric acid group-containing compound, or a salt thereof.</u><u style="single">(Item 32)</u><u style="single">The method according to any one of items 1 to 5, wherein the reporter moiety does not substantially sub-fragment under the conditions used to measure the analyte.</u><u style="single">(Item 33)</u><u style="single">The method according to any one of items 2 to 5, wherein the reporter moiety is not a biopolymer.</u><u style="single">(Item 34)</u><u style="single">The method according to any one of items 2 to 5, wherein the reporter moiety is not a polymer.</u><u style="single">(Item 35)</u><u style="single">The method according to any one of items 1 to 5, wherein the linker is a carbonyl or thiocarbonyl group.</u><u style="single">(Item 36)</u><u style="single">The method according to any one of items 1 to 4, wherein the linker is a polymer or biopolymer moiety.</u><u style="single">(Item 37)</u><u style="single">36. The method of item 36, wherein the polymer moiety can be sub-fragmented.</u><u style="single">(Item 38)</u><u style="single">The method according to any one of items 1 to 5, wherein each of the one or more differentially labeled analytes comprises an isomeric label that identifies the sample that results in the differentially labeled analyte.</u><u style="single">(Item 39)</u><u style="single">The method according to any one of items 1 to 5, wherein each of the one or more differentially labeled analytes comprises an isobaric label that identifies the sample that results in the differentially labeled analyte.</u><u style="single">(Item 40)</u><u style="single">Each isotope-labeled analysis has a cyclic nitrogen atom that is N-alkylated by a substituted or unsubstituted acetic acid moiety to which the analyte is attached via the carbonyl carbon of the N-alkylacetic acid moiety 5,6. , Or the method of item 39, wherein the 7-membered heterocycle, each with a different label containing one or more heavy atom isotopes.</u><u style="single">(Item 41)</u><u style="single">Each of the isobaric labeled analytes in the sample mixture has the following formula:</u><chemistry num="2"><img file="JP2010190902A_D0004.tif" /></chemistry><u style="single">Including</u><u style="single"> During the ceremony</u><u style="single"> a) Z is O, S, NH, or NR</u>^{<u style="single">1</u>}<u style="single">And</u><u style="single"> b) Each J is the same or different, H, deuterium (D), R</u>^{<u style="single">1</u>}<u style="single">, OR</u>^{<u style="single">1</u>}<u style="single">, SR</u>^{<u style="single">1</u>}<u style="single">, NHR</u>^{<u style="single">1</u>}<u style="single">, N (R)</u>^{<u style="single">1</u>}<u style="single">)</u>_{<u style="single">2</u>}<u style="single">, Fluorine, chlorine, bromine, or iodine,</u><u style="single">c) W is an atom or group located in the cyclic nitrogen in ortho, meta, or para, NH, NR.</u>^{<u style="single">1</u>}<u style="single">, NR</u>^{<u style="single">2</u>}<u style="single">, PR</u>^{<u style="single">1</u>}<u style="single">, PR</u>^{<u style="single">2</u>}<u style="single">, O, or S,</u><u style="single">d) Each carbon of the above heterocycle is given by the formula CJ.</u>_{<u style="single">2</u>}<u style="single">Have,</u><u style="single">e) Each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is , Separately containing bonded hydrogen, deuterium, and / or fluorine atoms,</u><u style="single">f) R</u>^{<u style="single">2</u>}<u style="single">Is a cleavable linker that cleaves an aminoalkyl, hydroxyalkyl, thioalkyl group, or the above reagent to a solid support, wherein the aminoalkyl, hydroxyalkyl, or thioalkyl group is 1 to 8 pieces. It has a carbon atom of, and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, and the carbon atoms of the alkyl and aryl groups are separately bonded hydrogen, deuterium, and / or fluorine. The method of item 40, which comprises an atom.</u><u style="single">(Item 42)</u><u style="single">40. The method of item 40, wherein the isobaric labeled analyte is a peptide.</u><u style="single">(Item 43)</u><u style="single">The above sample mixture has the following formula:</u><chemistry num="3"><img file="JP2010190902A_D0005.tif" /></chemistry><u style="single">41. The method of item 41, comprising one or more isobaric labeling analytes of.</u><u style="single">(Item 44)</u><u style="single">The above sample mixture has the following formula:</u><chemistry num="4"><img file="JP2010190902A_D0006.tif" /></chemistry><u style="single">41. The method of item 41, comprising one or more isobaric labeling analytes of.</u><u style="single">(Item 45)</u><u style="single">The above sample mixture has the following formula:</u><chemistry num="5"><img file="JP2010190902A_D0007.tif" /></chemistry><u style="single">Includes one or more isobaric labeling analytes of</u><u style="single"> In the formula, each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is 41. The method of item 41, which comprises, separately, bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">(Item 46)</u><u style="single">The above sample mixture has the following formula:</u><chemistry num="6"><img file="JP2010190902A_D0008.tif" /></chemistry><u style="single">Includes one or more isobaric labeling analytes of</u><u style="single"> During the ceremony</u><u style="single">a) G'is an aminoalkyl, hydroxyalkyl, thioalkyl group having 1 to 8 carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the alkyl and aryl groups are described above. Carbon atoms separately contain bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">b) Each carbon of the above heterocycle has the formula CJ</u>_{<u style="single">2</u>}<u style="single">Where each J is the same or different, H, deuterium (D), R</u>^{<u style="single">1</u>}<u style="single">, OR</u>^{<u style="single">1</u>}<u style="single">, SR</u>^{<u style="single">1</u>}<u style="single">, NHR</u>^{<u style="single">1</u>}<u style="single">, N (R)</u>^{<u style="single">1</u>}<u style="single">)</u>_{<u style="single">2</u>}<u style="single">, Fluorine, Chlorine, Bromine, and Iodine.</u><u style="single">c) Each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is 41. The method of item 41, which comprises, separately, bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">(Item 47)</u><u style="single">Each of the isobaric labeled analytes in the sample mixture has the following formula:</u><chemistry num="7"><img file="JP2010190902A_D0009.tif" /></chemistry><u style="single">Including</u><u style="single"> During the ceremony</u><u style="single"> a) Z is O, S, NH, or NR</u>^{<u style="single">1</u>}<u style="single">And</u><u style="single"> b) Each J is the same or different, H, deuterium (D), R</u>^{<u style="single">1</u>}<u style="single">, OR</u>^{<u style="single">1</u>}<u style="single">, SR</u>^{<u style="single">1</u>}<u style="single">, NHR</u>^{<u style="single">1</u>}<u style="single">, N (R)</u>^{<u style="single">1</u>}<u style="single">)</u>_{<u style="single">2</u>}<u style="single">, Fluorine, Chlorine, Bromine, and Iodine.</u><u style="single">c) Each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is 39. The method of item 39, which comprises, separately, bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">(Item 48)</u><u style="single">47. The method of item 47, wherein the isobaric labeling analyte is a peptide.</u><u style="single">(Item 49)</u><u style="single">The above sample mixture has the following formula:</u><chemistry num="8"><img file="JP2010190902A_D0010.tif" /></chemistry><u style="single">Includes one or more isobaric labeling analytes of</u><u style="single"> In the formula, each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is 47. The method of item 47, which comprises, separately, bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">(Item 50)</u><u style="single">Each of the different labeling reagents in the above set is support binding and is bound and bound to the support via a cleavable linker to react each different sample with a support holding the different labeling reagents. Moreover, the above method is performed before the step (b) is executed.</u><u style="single"> i) The step of optionally washing the resin to remove components of the sample that do not react with the reactive groups of the labeling reagent;</u><u style="single"> ii) A step of cleaving the cleaveable linker, wherein each sample collects two or more kinds of differentially labeled samples containing one or more kinds of labeled analytes.</u><u style="single"> Further include</u><u style="single"> The method of any of items 1-5, wherein the labeled analyte associated with a particular sample is identifiable and / quantifiable by the unique reporter bound thereto.</u><u style="single">(Item 51)</u><u style="single">Each different labeling reagent in the above set has the following formula:</u><u style="single"> EF-RP-X-LK-Y-RG</u><u style="single">Is a solid support of</u><u style="single"> During the ceremony</u><u style="single"> i) RG is a reactive group that is a nucleophile or electrophile, and is capable of reacting with one or more of the above-mentioned reactive analysts of the above-mentioned sample.</u><u style="single"> ii) The RP is a reporter moiety that contains a fixed charge or is capable of being ionized, where the mass of each reporter shall be different for each reagent in the above set.</u><u style="single"> iii) LK is a linker moiety that binds the reaction group and the reporter group, and the mass of the linker is the integrated total mass of the combination of the reporter and the linker with respect to each reagent in the set. Correct the difference in total mass between the reporters for the different labeling reagents in the above set so that they are the same.</u><u style="single"> iv) X is the bond between the reporter atom and the linker,</u><u style="single"> v) Y is the bond between the atom of the linker and the atom of the reactive group, where when the labeling reagent reacts with the reactive analyte, the bond Y attaches the linker to the analyte. Combine,</u><u style="single"> vi) Bonds X and Y are fragmented at least in part of the labeled analyte when subjected to dissociation energy levels.</u><u style="single"> vii) E is a solid support,</u><u style="single"> viii) The method of item 50, wherein F is a cleavable linker that binds to the solid support and cleavably to the reporter.</u><u style="single">(Item 52)</u><u style="single">The above set of labeling reagents is one or more of the following support binding labeling reagents, i.e.</u><chemistry num="9"><img file="JP2010190902A_D0011.tif" /></chemistry><u style="single">Including</u><u style="single"> During the ceremony</u><u style="single"> i) RG is a reactive group that is a nucleophile or electrophile, and is capable of reacting with one or more of the above-mentioned reactive analysts of the above-mentioned sample.</u><u style="single"> ii) E is a solid support,</u><u style="single"> iii) F is a cleavable linker that binds to the solid support and cleavably to the reporter.</u><u style="single"> iv) G is an aminoalkyl, hydroxyalkyl, or thioalkyl group that is cleaveable to the cleavable linker, wherein the aminoalkyl, hydroxyalkyl, or thioalkyl group is 1 to 8 carbon atoms. And optionally contain a heteroatom or a substituted or unsubstituted aryl group, further, the carbon atoms of the alkyl and aryl groups separately contain bonded hydrogen, deuterium, and / or fluorine atoms. ,</u><u style="single"> v) Each carbon of the above heterocycle is given by the formula CJ.</u>_{<u style="single">2</u>}<u style="single">Where each J is the same or different, H, deuterium (D), R</u>^{<u style="single">1</u>}<u style="single">, OR</u>^{<u style="single">1</u>}<u style="single">, SR</u>^{<u style="single">1</u>}<u style="single">, NHR</u>^{<u style="single">1</u>}<u style="single">, N (R)</u>^{<u style="single">1</u>}<u style="single">)</u>_{<u style="single">2</u>}<u style="single">, Fluorine, Chlorine, Bromine, and Iodine.</u><u style="single"> vi) Each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is 51. The method of item 51, which comprises, separately, bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">(Item 53)</u><u style="single">The method of item 50, wherein the support is composed of polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, polyacrylamide, glass, silica, controlled porous glass (CPG), or reverse phase silica.</u><u style="single">(Item 54)</u><u style="single">The method of item 50, wherein the solid support is beads, spheres, particles, granules, gels, membranes, or planar.</u><u style="single">(Item 55)</u><u style="single">the following:</u><u style="single"> c) Any of items 1 to 5, further comprising the step of digesting each sample with at least one enzyme prior to performing step a) to partially or completely degrade the components of the sample. The method described in.</u><u style="single">(Item 56)</u><u style="single">55. The method of item 55, wherein the enzyme is a proteolytic enzyme.</u><u style="single">(Item 57)</u><u style="single">56. The method of item 56, wherein the proteolytic enzyme is trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin, or carboxypeptidase C.</u><u style="single">(Item 58)</u><u style="single">The above method is as follows:</u><u style="single"> c) Step of separating the above sample mixture,</u><u style="single">The method according to any one of items 1 to 5, further comprising.</u><u style="single">(Item 59)</u><u style="single">58. The method of item 58, wherein the separation is performed by chromatography.</u><u style="single">(Item 60)</u><u style="single">59. The method of item 59, wherein the method of chromatographic separation is normal phase chromatography, reverse phase chromatography, ion exchange chromatography, size exclusion chromatography, or affinity chromatography.</u><u style="single">(Item 61)</u><u style="single">58. The method of item 58, wherein the separation is performed by electrophoresis.</u><u style="single">(Item 62)</u><u style="single">61. The method of item 61, wherein the electrophoresis separation is one-dimensional electrophoresis separation, two-dimensional electrophoresis separation, or capillary electrophoresis separation.</u><u style="single">(Item 63)</u><u style="single">The above method is as follows:</u><u style="single">c) Prior to performing step a), each sample is digested with at least one enzyme to partially or completely degrade the components of the sample; and</u><u style="single">d) Step of separating the above sample mixture,</u><u style="single"> The method according to any one of items 1 to 5, further comprising.</u><u style="single">(Item 64)</u><u style="single">63. The method of item 63, wherein the enzyme is a proteolytic enzyme.</u><u style="single">(Item 65)</u><u style="single">The method of item 64, wherein the proteolytic enzyme is trypsin, papain, pepsin, chymotrypsin, or carboxypeptidase C.</u><u style="single">(Item 66)</u><u style="single">63. The method of item 63, wherein the separation is performed by chromatography.</u><u style="single">(Item 67)</u><u style="single">66. The method of item 66, wherein the method of chromatographic separation is normal phase chromatography, reverse phase chromatography, ion exchange chromatography, size exclusion chromatography, or affinity chromatography.</u><u style="single">(Item 68)</u><u style="single">63. The method of item 63, wherein the separation is electrophoretic separation.</u><u style="single">(Item 69)</u><u style="single">68. The method of item 68, wherein the electrophoresis separation is one-dimensional electrophoresis separation, two-dimensional electrophoresis separation, or capillary electrophoresis separation.</u><u style="single">(Item 70)</u><u style="single">7. The method of item 7, wherein the identity of the labeled analyte in relation to the selected mass relative to the charge ratio is measured by analysis of the daughter fragment ions.</u><u style="single">(Item 71)</u><u style="single">The method of item 70, wherein the relative amount of each reporter in the second mass spectrometry is measured relative to the other reporter.</u><u style="single">(Item 72)</u><u style="single">Two or more of the above combined to form the mixture by correlating the relative amount of each reporter associated with the identified analyte with the amount of each sample added to form the sample mixture. 71. The method of item 71, wherein the relative amount of the analyte in each of the samples is measured.</u><u style="single">(Item 73)</u><u style="single"> (i) The sample mixture contains a known amount of calibration criteria for the identified analyte, and the absolute amount of each reporter is measured relative to the amount of reporter associated with the calibration criteria.</u><u style="single"> (ii) The method of item 72, wherein the absolute amount of the identified analyte in each different sample of the sample mixture is measured relative to the amount of each reporter.</u><u style="single">(Item 74)</u><u style="single">The above two types combined to form the sample mixture by repeating steps (d) to (f) one or more times on the ions of the labeled analyte selected with different selected masses to the charge ratio. 72. The method of item 72, further comprising identifying and / or measuring the relative amount of one or more other analytes in each of the above samples.</u><u style="single">(Item 75)</u><u style="single">The above two types combined to form the sample mixture by repeating steps (d) to (f) one or more times on the ions of the labeled analyte selected with different selected masses to the charge ratio. The method of item 73, further comprising identifying and / or measuring the absolute amount of one or more other analytes in each of the above samples.</u><u style="single">(Item 76)</u><u style="single">The identity and relative amount of one or more proteins in each of the two or more samples that the analyte is a peptide and is combined to form the sample mixture is to form the sample mixture. 72. The method of item 72, which is measured based on the identity and relative amount of the one or more peptides in each of the two or more samples to be combined.</u><u style="single">(Item 77)</u><u style="single">The identity and relative amount of one or more proteins in each of the two or more samples that the analyte is a peptide and is combined to form the sample mixture is to form the sample mixture. 73. The method of item 73, which is measured based on the identity and absolute amount of the one or more peptides in each of the two or more samples to be combined.</u><u style="single">(Item 78)</u><u style="single">the following:</u><u style="single">c) Prior to performing step (a), each sample is digested with at least one enzyme to partially or completely degrade the components of the sample; and</u><u style="single">d) The step of separating the sample mixture before performing step (c),</u><u style="single"> 7. The method of item 7.</u><u style="single">(Item 79)</u><u style="single">The method of item 78, wherein the enzyme is a proteolytic enzyme.</u><u style="single">(Item 80)</u><u style="single">79. The method of item 79, wherein the proteolytic enzyme is trypsin, papain, pepsin, chymotrypsin, or carboxypeptidase C.</u><u style="single">(Item 81)</u><u style="single">The method of item 78, wherein the separation is performed by chromatography.</u><u style="single">(Item 82)</u><u style="single">81. The method of item 81, wherein the method of chromatographic separation is normal phase chromatography, reverse phase chromatography, ion exchange chromatography, size exclusion chromatography, or affinity chromatography.</u><u style="single">(Item 83)</u><u style="single">The method of item 78, wherein the separation is electrophoretic separation.</u><u style="single">(Item 84)</u><u style="single">The method according to item 83, wherein the electrophoresis separation is one-dimensional electrophoresis separation, two-dimensional electrophoresis separation, or capillary electrophoresis separation.</u><u style="single">(Item 85)</u><u style="single">58. The method of item 78, wherein the identity of the labeled analyte in relation to the selected mass relative to the charge ratio is measured by analysis of the daughter fragment ions.</u><u style="single">(Item 86)</u><u style="single">The method of item 85, wherein the relative amount of each reporter in the second mass spectrometry is measured relative to the other reporter.</u><u style="single">(Item 87)</u><u style="single">Two or more of the above combined to form the mixture by correlating the relative amount of each reporter associated with the identified analyte with the amount of each sample added to form the sample mixture. 86. The method of item 86, wherein the relative amount of the analyte in each of the samples of.</u><u style="single">(Item 88)</u><u style="single"> (i) The sample mixture contains a known amount of calibration criteria for the identified analyte, and the absolute amount of each reporter is measured relative to the amount of reporter associated with the calibration criteria.</u><u style="single"> (ii) The method of item 87, wherein the absolute amount of the identified analyte in each different sample of the sample mixture is measured relative to the amount of each reporter.</u><u style="single">(Item 89)</u><u style="single">The above two types combined to form the sample mixture by repeating steps (d) to (f) one or more times on the ions of the labeled analyte selected with different selected masses to the charge ratio. 8. The method of item 87, further comprising identifying and / or measuring the relative amount of one or more other analytes in each of the above samples.</u><u style="single">(Item 90)</u><u style="single">The above two types combined to form the sample mixture by repeating steps (d) to (f) one or more times on the ions of the labeled analyte selected with different selected masses to the charge ratio. 88. The method of item 88, further comprising identifying and / or measuring the absolute amount of one or more other analytes in each of the above samples.</u><u style="single">(Item 91)</u><u style="single">The identity and relative amount of one or more proteins in each of the two or more samples that the analyte is a peptide and is combined to form the sample mixture is to form the sample mixture. 87. The method of item 87, which is measured based on the identity and relative amount of the one or more peptides in each of the two or more samples to be combined.</u><u style="single">(Item 92)</u><u style="single">The identity and relative amount of one or more proteins in each of the two or more samples that the analyte is a peptide and is combined to form the sample mixture is to form the sample mixture. 88. The method of item 88, which is measured based on the identity and absolute amount of the one or more peptides in each of the two or more samples to be combined.</u><u style="single">(Item 93)</u><u style="single">A mixture containing at least two labeled analytes, each of which originated from a different sample combined to form the mixture, each of which has the following formula:</u><u style="single"> RP-X-LK-Y-Analyst</u><u style="single">Or contains its salt,</u><u style="single"> During the ceremony</u><u style="single">a) The RP is a reporter moiety that contains a fixed charge or can be ionized, where the mass of each reporter shall be different for each sample.</u><u style="single">b) LK is the linker moiety that binds the analyte to the reporter's group, where the mass of the linker is the integrated total mass of the combination of the reporter and the linker for each labeled analyte. Correct for the difference in total mass between the different reporters above so that they are the same,</u><u style="single">c) X is the bond between the reporter atom and the linker,</u><u style="single">d) Y is the bond between the atom of the linker and the atom of the analyte.</u><u style="single">e) Bonds X and Y are fragmented at least in part of the labeled analyte when subjected to dissociation energy levels.</u><u style="single">Where RP is</u><u style="single"> i) Have a total mass of less than 250 daltons and / or</u><u style="single"> ii) Substantially no sub-fragmentation and / or under the conditions of dissociation energy applied to cause at least some fragmentation of the bond X and Y of the labeled analyte on a mass spectrometer. ,</u><u style="single"> iii) A mixture that is not a polymer or a biopolymer.</u><u style="single">(Item 94)</u><u style="single">A mixture containing at least two labeled analytes, each of which originated from a different sample combined to form the mixture, each of which has the following formula:</u><u style="single"> RP-X-LK-Y-Contains analyte or salt thereof,</u><u style="single"> During the ceremony</u><u style="single">a) The RP is a reporter moiety that contains a fixed charge or can be ionized, where the mass of each reporter shall be different for each sample.</u><u style="single">b) LK is the linker moiety that binds the analyte to the reporter's group, where the mass of the linker is the integrated total mass of the combination of the reporter and the linker for each labeled analyte. Correct for the difference in total mass between the different reporters above so that they are the same,</u><u style="single">c) X is the bond between the reporter atom and the linker,</u><u style="single">d) Y is the bond between the atom of the linker and the atom of the analyte.</u><u style="single">e) Bonds X and Y are fragmented at least in part of the labeled analyte when subjected to dissociation energy levels.</u><u style="single"> Here, the linker LK is a mixture that undergoes neutral loss under the conditions of applied dissociation energy that causes bond X and Y fragmentation on a mass spectrometer.</u><u style="single">(Item 95)</u><u style="single">A mixture containing at least two labeled analytes, each of which originated from a different sample combined to form the mixture, each of which has the following formula:</u><u style="single"> RP-X-LK-Y-Analyst</u><u style="single">Or contains its salt,</u><u style="single"> During the ceremony</u><u style="single">a) The RP is a reporter moiety that contains a fixed charge or can be ionized, where the mass of each reporter shall be different for each sample.</u><u style="single">b) LK is the linker moiety that binds the analyte to the reporter's group, where the mass of the linker is the integrated total mass of the combination of the reporter and the linker for each labeled analyte. Correct for the difference in total mass between the different reporters above so that they are the same,</u><u style="single">c) X is the bond between the reporter atom and the linker,</u><u style="single">d) Y is the bond between the atom of the linker and the atom of the analyte.</u><u style="single">e) Bonds X and Y are fragmented at least in part of the labeled analyte when subjected to dissociation energy levels.</u><u style="single"> Here, under the conditions of dissociation energy applied by a mass spectrometer, one fragmentation of bond X or Y induces fragmentation of the other of bond X or Y, a mixture.</u><u style="single">(Item 96)</u><u style="single">A mixture containing at least two labeled analytes, each of which originated from a different sample combined to form the mixture, each of which has the following formula:</u><u style="single"> RP-X-LK-Y-Analyst</u><u style="single">Or contains its salt,</u><u style="single"> During the ceremony</u><u style="single">a) The RP is a reporter moiety that contains a fixed charge or can be ionized, where the mass of each reporter shall be different for each sample.</u><u style="single">b) LK is the linker moiety that binds the analyte to the reporter's group, where the mass of the linker is the integrated total mass of the combination of the reporter and the linker for each labeled analyte. Correct for the difference in total mass between the different reporters above so that they are the same,</u><u style="single">c) X is the bond between the reporter atom and the linker,</u><u style="single">d) Y is the bond between the atom of the linker and the atom of the analyte.</u><u style="single">e) Bonds X and Y are fragmented at least in part of the labeled analyte when subjected to dissociation energy levels.</u><u style="single">here,</u><u style="single">i) Under the conditions of the dissociation energy applied by the mass spectrometer, bond X is less prone to fragmentation than bond Y and / or</u><u style="single">ii) Under the conditions of the dissociation energy applied by the mass spectrometer, the bound X is less prone to fragmentation than the peptide bond of the Z-pro amino acid dimer or the Z-asp amino acid dimer, where Z is A mixture characterized by being any natural amino acid, pro being proline and asp being aspartic acid.</u><u style="single">(Item 97)</u><u style="single">A mixture containing at least two labeled analytes, each of which originated from a different sample combined to form the mixture, each of which has the following formula:</u><u style="single"> RP-X-LK-Y-Analyst</u><u style="single">Or contains its salt,</u><u style="single"> During the ceremony</u><u style="single">a) The RP is a reporter moiety that contains a fixed charge or can be ionized, where the mass of each reporter shall be different for each sample.</u><u style="single">b) LK is the linker moiety that binds the reactive group to the reporter group, where</u><u style="single">i) The mass of the linker is the total mass between the reporters for the different labeling reagents in the set so that the integrated total mass of the combination of the reporter and the linker is the same for each reagent in the set. Correct the difference and</u><u style="single">ii) The above linker contains at least one heavy atom isotope and has the following formula:</u><chemistry num="10"><img file="JP2010190902A_D0012.tif" /></chemistry><u style="single">Have,</u><u style="single"> In the formula, R</u>^{<u style="single">1</u>}<u style="single">Is the same or different, and is an alkyl group containing 1 to 8 carbon atoms, and the alkyl group may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the alkyl group is described herein. And the carbon atoms of the aryl group separately contain bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">c) X is the bond between the reporter atom and the linker,</u><u style="single">d) Y is a mixture characterized by being a bond between the atom of the linker and the atom of the analyte.</u><u style="single">(Item 98)</u><u style="single">The mixture according to any one of items 93 to 97, wherein the one or more analytes are peptides.</u><u style="single">(Item 99)</u><u style="single">The mixture according to any one of items 93 to 97, wherein the one or more analytes are proteins.</u><u style="single">(Item 100)</u><u style="single">The mixture according to any one of items 93 to 97, wherein the one or more kinds of the above-mentioned analytes are nucleic acid molecules.</u><u style="single">(Item 101)</u><u style="single">The mixture according to any one of items 93 to 97, wherein the reporter is a substituted or unsubstituted morpholine, piperidine, or piperazine compound, or a salt thereof.</u><u style="single">(Item 102)</u><u style="single">The mixture according to any one of items 93 to 97, wherein the reporter is a carboxylic acid, a sulfonic acid, or a phosphoric acid group-containing compound, or a salt thereof.</u><u style="single">(Item 103)</u><u style="single">The mixture according to any one of items 93 to 97, wherein the linker is a carbonyl or thiocarbonyl group.</u><u style="single">(Item 104)</u><u style="single">The mixture according to any one of items 93 to 97, wherein each of the at least two labeled analytes comprises an isomeric label.</u><u style="single">(Item 105)</u><u style="single">The mixture according to any one of items 93 to 97, wherein each of the at least two labeled analytes comprises an isobaric label.</u><u style="single">(Item 106)</u><u style="single">Each of the at least two labeled analysts has a cyclic nitrogen atom that is N-alkylated by a substituted or unsubstituted acetic acid moiety to which the analyte is attached via the carbonyl carbon of the N-alkylacetic acid moiety 5,6. , Or the mixture of item 105, comprising an isobaric label that is a 7-membered heterocycle, each of which contains one or more heavy atom isotopes.</u><u style="single">(Item 107)</u><u style="single">Each of the at least two isobaric labeled analytes in the mixture has the following formula:</u><chemistry num="11"><img file="JP2010190902A_D0013.tif" /></chemistry><u style="single">Including</u><u style="single"> During the ceremony</u><u style="single"> a) Z is O, S, NH, or NR</u>^{<u style="single">1</u>}<u style="single">And</u><u style="single"> b) Each J is the same or different, H, deuterium (D), R</u>^{<u style="single">1</u>}<u style="single">, OR</u>^{<u style="single">1</u>}<u style="single">, SR</u>^{<u style="single">1</u>}<u style="single">, NHR</u>^{<u style="single">1</u>}<u style="single">, N (R)</u>^{<u style="single">1</u>}<u style="single">)</u>_{<u style="single">2</u>}<u style="single">, Fluorine, chlorine, bromine, or iodine,</u><u style="single">c) W is an atom or group located in the cyclic nitrogen in ortho, meta, or para, NH, NR.</u>^{<u style="single">1</u>}<u style="single">, NR</u>^{<u style="single">2</u>}<u style="single">, PR</u>^{<u style="single">1</u>}<u style="single">, PR</u>^{<u style="single">2</u>}<u style="single">, O, or S,</u><u style="single">d) Each carbon of the above heterocycle is given by the formula CJ.</u>_{<u style="single">2</u>}<u style="single">Have,</u><u style="single">e) Each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is , Separately containing bonded hydrogen, deuterium, and / or fluorine atoms,</u><u style="single">f) R</u>^{<u style="single">2</u>}<u style="single">Is a cleavable linker that cleaves an aminoalkyl, hydroxyalkyl, thioalkyl group, or the above reagent to a solid support, wherein the aminoalkyl, hydroxyalkyl, or thioalkyl group is 1 to 8 pieces. It has a carbon atom of, and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, and the carbon atoms of the alkyl and aryl groups are separately bonded hydrogen, deuterium, and / or fluorine. The mixture according to item 106, which comprises an atom.</u><u style="single">(Item 108)</u><u style="single">The above mixture has the following formula:</u><chemistry num="12"><img file="JP2010190902A_D0014.tif" /></chemistry><u style="single">107. The mixture according to item 107, comprising one or more isobaric labeling analytes of.</u><u style="single">(Item 109)</u><u style="single">The above mixture has the following formula:</u><chemistry num="13"><img file="JP2010190902A_D0015.tif" /></chemistry><u style="single">107. The mixture according to item 107, comprising one or more isobaric labeling analytes of.</u><u style="single">(Item 110)</u><u style="single">The above mixture has the following formula:</u><chemistry num="14"><img file="JP2010190902A_D0016.tif" /></chemistry><u style="single">Includes one or more isobaric labeling analytes of</u><u style="single"> During the ceremony</u><u style="single">a) G'is an aminoalkyl, hydroxyalkyl, or thioalkyl group having 1 to 8 carbon atoms, optionally containing a heteroatom or a substituted or unsubstituted aryl group, wherein the alkyl and aryl groups are described above. Carbon atoms separately contain bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">b) Each carbon of the above heterocycle has the formula CJ</u>_{<u style="single">2</u>}<u style="single">Where each J is the same or different, H, deuterium (D), R</u>^{<u style="single">1</u>}<u style="single">, OR</u>^{<u style="single">1</u>}<u style="single">, SR</u>^{<u style="single">1</u>}<u style="single">, NHR</u>^{<u style="single">1</u>}<u style="single">, N (R)</u>^{<u style="single">1</u>}<u style="single">)</u>_{<u style="single">2</u>}<u style="single">, Fluorine, Chlorine, Bromine, and Iodine.</u><u style="single">c) Each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is The mixture according to item 107, which comprises, separately, bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">(Item 111)</u><u style="single">The above mixture has the following formula:</u><chemistry num="15"><img file="JP2010190902A_D0017.tif" /></chemistry><u style="single">Includes one or more isobaric labeling analytes of</u><u style="single"> During the ceremony</u><u style="single"> a) Z is O, S, NH, or NR</u>^{<u style="single">1</u>}<u style="single">And</u><u style="single"> b) Each J is the same or different, H, deuterium (D), R</u>^{<u style="single">1</u>}<u style="single">, OR</u>^{<u style="single">1</u>}<u style="single">, SR</u>^{<u style="single">1</u>}<u style="single">, NHR</u>^{<u style="single">1</u>}<u style="single">, N (R)</u>^{<u style="single">1</u>}<u style="single">)</u>_{<u style="single">2</u>}<u style="single">, Fluorine, Chlorine, Bromine, and Iodine.</u><u style="single">c) Each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is The mixture according to item 105, which comprises, separately, bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">(Item 112)</u><u style="single">The mixture according to any of items 93 to 97, further comprising one or more calibration criteria.</u><u style="single">(Item 113)</u><u style="single">An active ester that is a 5, 6, or 7-membered heterocycle with a cyclic nitrogen atom that is N-alkylated by a substituted or unsubstituted acetic acid moiety to which the alcohol moiety of the active ester is attached via the carbonyl carbon of the N-alkyl acetic acid moiety. A compound characterized in that the above-mentioned compound is isotically enriched with one or more kinds of heavy atom isotopes.</u><u style="single">(Item 114)</u><u style="single">The compound according to item 113, wherein the compound is isotopically enriched with three or more types of heavy atom isotopes.</u><u style="single">(Item 115)</u><u style="single">The compound according to item 113, wherein the heterocycle is substituted with one or more substituents.</u><u style="single">(Item 116)</u><u style="single">The compound according to item 115, wherein the one or more substituents are alkyl, alkoxy, or aryl groups.</u><u style="single">(Item 117)</u><u style="single">The compound according to item 116, wherein the one or more substituents are a protected or unprotected amine group, hydroxyl group, or thiol group.</u><u style="single">(Item 118)</u><u style="single">The compound according to item 113, wherein the heterocycle is an aliphatic compound.</u><u style="single">(Item 119)</u><u style="single">The compound according to item 113, wherein the heterocycle is aromatic.</u><u style="single">(Item 120)</u><u style="single">The compound according to item 113, wherein the heterocycle comprises one or more additional nitrogen, oxygen, or sulfur atoms.</u><u style="single">(Item 121)</u><u style="single">The compound according to item 113, wherein the active ester is an N-hydroxysuccinimide ester.</u><u style="single">(Item 122)</u><u style="single">The compound according to item 113, wherein the compound is a salt.</u><u style="single">(Item 123)</u><u style="single">The compound according to item 113, wherein the compound is a mono TFA salt, a mono HCl salt, a bis TFA salt, or a bis HCl salt.</u><u style="single">(Item 124)</u><u style="single">The compound of item 113, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 80%.</u><u style="single">(Item 125)</u><u style="single">The compound of item 113, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 93%.</u><u style="single">(Item 126)</u><u style="single">The compound of item 113, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 96%.</u><u style="single">(Item 127)</u><u style="single">The following formula:</u><chemistry num="16"><img file="JP2010190902A_D0018.tif" /></chemistry><u style="single">N-substituted morpholine acetic acid active ester compound or a salt thereof,</u><u style="single"> During the ceremony</u><u style="single"> LG is a leaving group for active esters,</u><u style="single"> X is O or S,</u><u style="single"> Each Z is a linear or branched C1 to C6 alkyl group that may separately contain hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino acid side chains, or optionally substituted or unsubstituted aryl groups. Yes, where the carbon atoms of the alkyl or aryl group each separately contain a bonded hydrogen, deuterium, or fluorine atom.</u><u style="single"> Here, a compound characterized in that the above N-substituted morpholine acetic acid active ester is isotopically enriched with one or more kinds of heavy atom isotopes.</u><u style="single">(Item 128)</u><u style="single">The compound according to item 127, wherein the N-substituted morpholine acetic acid active ester is enriched with three or more heavy atom isotopes.</u><u style="single">(Item 129)</u><u style="single">LG</u><chemistry num="17"><img file="JP2010190902A_D0019.tif" /></chemistry><u style="single">The compound according to item 127, wherein X is O or S.</u><u style="single">(Item 130)</u><u style="single">The compound according to item 127, wherein LG is N-hydroxysuccinimide.</u><u style="single">(Item 131)</u><u style="single">The compound according to item 127, wherein each Z is separately hydrogen, deuterium, fluorine, chlorine, bromine, or iodine.</u><u style="single">(Item 132)</u><u style="single">The compound according to item 127, wherein each Z is separately hydrogen, methyl, or methoxy.</u><u style="single">(Item 133)</u><u style="single">X is</u>^{<u style="single">16</u>}<u style="single">O or</u>^{<u style="single">18</u>}<u style="single">The compound according to item 127, which is O.</u><u style="single">(Item 134)</u><u style="single">The nitrogen atom of the morpholine ring</u>^{<u style="single">14</u>}<u style="single">N or</u>^{<u style="single">15</u>}<u style="single">The compound according to item 127, which is N.</u><u style="single">(Item 135)</u><u style="single">The following formula:</u><chemistry num="18"><img file="JP2010190902A_D0020.tif" /></chemistry><u style="single">It is the above compound of</u><u style="single"> During the ceremony</u><u style="single"> Each C</u>^{<u style="single">*</u>}<u style="single">Separately</u>^{<u style="single">12</u>}<u style="single">C or</u>^{<u style="single">13</u>}<u style="single">C and</u><u style="single"> LG is a leaving group for active esters,</u><u style="single"> X is O or S,</u><u style="single"> Each Z is a linear or branched C1 to C6 alkyl group that may separately contain hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino acid side chains, or optionally substituted or unsubstituted aryl groups. The compound according to item 127, wherein the carbon atom of the alkyl or aryl group is each separately comprising a bonded hydrogen, deuterium, or fluorine atom.</u><u style="single">(Item 136)</u><u style="single">The compound according to item 127, wherein the compound is a mono TFA salt or a mono HCl salt.</u><u style="single">(Item 137)</u><u style="single">The compound of item 127, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 80%.</u><u style="single">(Item 138)</u><u style="single">The compound of item 127, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 93%.</u><u style="single">(Item 139)</u><u style="single">The compound of item 127, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 96%.</u><u style="single">(Item 140)</u><u style="single">The following formula:</u><chemistry num="19"><img file="JP2010190902A_D0021.tif" /></chemistry><u style="single">N-substituted piperidine acetic acid active ester compound or a salt thereof,</u><u style="single"> During the ceremony</u><u style="single"> LG is a leaving group for active esters,</u><u style="single"> X is O or S,</u><u style="single"> Each Z is a linear or branched C1 to C6 alkyl group that may separately contain hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino acid side chains, or optionally substituted or unsubstituted aryl groups. Yes, where the carbon atoms of the alkyl or aryl group each separately contain a bonded hydrogen, deuterium, or fluorine atom.</u><u style="single"> Here, a compound characterized in that the above N-substituted piperidine acetic acid active ester is isotopically enriched with one or more kinds of heavy atom isotopes.</u><u style="single">(Item 141)</u><u style="single">The compound according to item 140, wherein the N-substituted piperidine acetic acid active ester is enriched with three or more heavy atom isotopes.</u><u style="single">(Item 142)</u><u style="single">LG</u><chemistry num="20"><img file="JP2010190902A_D0022.tif" /></chemistry><u style="single">The compound according to item 140, wherein X is O or S.</u><u style="single">(Item 143)</u><u style="single">The compound according to item 140, wherein LG is N-hydroxysuccinimide.</u><u style="single">(Item 144)</u><u style="single">The compound according to item 140, wherein each Z is separately hydrogen, deuterium, fluorine, chlorine, bromine, or iodine.</u><u style="single">(Item 145)</u><u style="single">The compound according to item 140, wherein each Z is separately hydrogen, methyl, or methoxy.</u><u style="single">(Item 146)</u><u style="single">X is</u>^{<u style="single">16</u>}<u style="single">O or</u>^{<u style="single">18</u>}<u style="single">The compound according to item 140, which is O.</u><u style="single">(Item 147)</u><u style="single">The nitrogen atom in the ring of piperidine</u>^{<u style="single">14</u>}<u style="single">N or</u>^{<u style="single">15</u>}<u style="single">The compound according to item 140, which is N.</u><u style="single">(Item 148)</u><u style="single">The following formula:</u><chemistry num="21"><img file="JP2010190902A_D0023.tif" /></chemistry><u style="single">It is the above compound of</u><u style="single"> During the ceremony</u><u style="single"> Each C</u>^{<u style="single">*</u>}<u style="single">Separately</u>^{<u style="single">12</u>}<u style="single">C or</u>^{<u style="single">13</u>}<u style="single">C and</u><u style="single"> LG is a leaving group for active esters,</u><u style="single"> X is O or S,</u><u style="single"> Each Z is a linear or branched C1 to C6 alkyl group that may separately contain hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino acid side chains, or optionally substituted or unsubstituted aryl groups. The compound according to item 140, wherein the carbon atom of the alkyl or aryl group is each separately comprising a bonded hydrogen, deuterium, or fluorine atom.</u><u style="single">(Item 149)</u><u style="single">The compound according to item 140, wherein the compound is a mono TFA salt or a mono HCl salt.</u><u style="single">(Item 150)</u><u style="single">The compound according to item 140, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 80%.</u><u style="single">(Item 151)</u><u style="single">The compound according to item 140, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 93%.</u><u style="single">(Item 152)</u><u style="single">The compound according to item 140, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 96%.</u><u style="single">(Item 153)</u><u style="single">The following formula:</u><chemistry num="22"><img file="JP2010190902A_D0024.tif" /></chemistry><u style="single">N-substituted piperazine acetic acid active ester compound or a salt thereof,</u><u style="single"> During the ceremony</u><u style="single"> LG is a leaving group for active esters,</u><u style="single"> X is O or S,</u><u style="single"> Each Z is a linear or branched C1 to C6 alkyl group that may separately contain hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino acid side chains, or optionally substituted or unsubstituted aryl groups. Yes, where the carbon atoms of the alkyl or aryl group each separately contain a bonded hydrogen, deuterium, or fluorine atom.</u><u style="single"> Here, a compound characterized in that the above N-substituted piperazine acetic acid active ester is isotopically enriched with one or more kinds of heavy atom isotopes.</u><u style="single">(Item 154)</u><u style="single">The compound according to item 153, wherein the N-substituted piperazine acetic acid active ester is enriched with three or more heavy atom isotopes.</u><u style="single">(Item 155)</u><u style="single">LG</u><chemistry num="23"><img file="JP2010190902A_D0025.tif" /></chemistry><u style="single">153. The compound according to item 153, wherein X is O or S.</u><u style="single">(Item 156)</u><u style="single">153. The compound of item 153, wherein LG is N-hydroxysuccinimide.</u><u style="single">(Item 157)</u><u style="single">153. The compound according to item 153, wherein each Z is separately hydrogen, deuterium, fluorine, chlorine, bromine, or iodine.</u><u style="single">(Item 158)</u><u style="single">153. The compound according to item 153, wherein each Z is separately hydrogen, methyl, or methoxy.</u><u style="single">(Item 159)</u><u style="single">X is</u>^{<u style="single">16</u>}<u style="single">O or</u>^{<u style="single">18</u>}<u style="single">The compound according to item 153, which is O.</u><u style="single">(Item 160)</u><u style="single">Each nitrogen atom in the above piperazine ring</u>^{<u style="single">14</u>}<u style="single">N or</u>^{<u style="single">15</u>}<u style="single">The compound according to item 153, which is N.</u><u style="single">(Item 161)</u><u style="single">The following formula:</u><chemistry num="24"><img file="JP2010190902A_D0026.tif" /></chemistry><u style="single">It is the above compound of</u><u style="single"> During the ceremony</u><u style="single"> Each C</u>^{<u style="single">*</u>}<u style="single">Separately</u>^{<u style="single">12</u>}<u style="single">C or</u>^{<u style="single">13</u>}<u style="single">C and</u><u style="single"> LG is a leaving group for active esters,</u><u style="single"> X is O or S,</u><u style="single"> Each Z is a linear or branched C1 to C6 alkyl group that may separately contain hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino acid side chains, or optionally substituted or unsubstituted aryl groups. 153. The compound according to item 153, wherein the carbon atom of the alkyl or aryl group separately comprises a bonded hydrogen, deuterium, or fluorine atom.</u><u style="single">(Item 162)</u><u style="single">153. The compound according to item 153, wherein the compound is a mono TFA salt, a mono HCl salt, a bis TFA salt, or a bis HCl salt.</u><u style="single">(Item 163)</u><u style="single">153. The compound of item 153, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 80%.</u><u style="single">(Item 164)</u><u style="single">153. The compound according to item 153, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 93%.</u><u style="single">(Item 165)</u><u style="single">153. The compound of item 153, wherein each of the heavy atomic isotopes incorporated is present with an isotope purity of at least 96%.</u><u style="single">(Item 166)</u><u style="single"> a) Each reagent in the set has the following formula:</u><u style="single"> RP-X-LK-Y-RG</u><u style="single"> Or with the above set of two or more reagents suitable for labeling an analyte containing its salt,</u><u style="single"> (During the ceremony,</u><u style="single">i) RG is a reactive group that is an electrophile and is capable of reacting with one or more of the above reactive analytes of the above sample.</u><u style="single">ii) The RP is a reporter moiety that contains a fixed charge or is capable of being ionized, where the mass of each reporter shall be different for each reagent in the above set.</u><u style="single">iii) LK is a linker moiety that binds the reaction group and the reporter group, and here,</u><u style="single">a) The mass of the linker is the total mass between the reporters for different labeling reagents in the set so that the integrated total mass of the combination of the reporter and the linker is the same for each reagent in the set. Correct the difference and</u><u style="single">b) The above linker contains at least one heavy atom isotope and has the following formula:</u><chemistry num="25"><img file="JP2010190902A_D0027.tif" /></chemistry><u style="single">Have,</u><u style="single"> In the formula, R</u>^{<u style="single">1</u>}<u style="single">Is the same or different, and is an alkyl group containing 1 to 8 carbon atoms, and the alkyl group may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the alkyl group is described herein. And the carbon atoms of the aryl group separately contain bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">iv) X is the bond between the reporter atom and the linker,</u><u style="single">v) Y is the bond between the atom of the linker and the atom of the reactive group, where when the labeling reagent reacts with the reactive analyte, the bond Y attaches the linker to the analyte. Combine. )</u><u style="single"> b) One or more reagents, containers, enzymes, buffers, or instructions,</u><u style="single"> Including, kit.</u><u style="single">(Item 167)</u><u style="single">166. The kit of item 166, wherein the kit comprises a proteolytic enzyme.</u><u style="single">(Item 168)</u><u style="single">166. The kit of item 166, wherein the reactive group of each reagent in the set is an active ester.</u><u style="single">(Item 169)</u><u style="single">The alcohol portion of the active ester is based on the following formula:</u><chemistry num="26"><img file="JP2010190902A_D0028.tif" /></chemistry><u style="single">The kit of item 168, wherein X is O or S in the formula.</u><u style="single">(Item 170)</u><u style="single">168. The kit of item 168, wherein the active ester is an N-hydroxysuccinimide ester.</u><u style="single">(Item 171)</u><u style="single">166. The kit of item 166, wherein the reporter is a substituted or unsubstituted morpholine, piperidine, or piperazine.</u><u style="single">(Item 172)</u><u style="single">166. The kit of item 166, wherein the reporter comprises a carboxylic acid, sulfonic acid, or phosphoric acid group.</u><u style="single">(Item 173)</u><u style="single">166. The kit of item 166, wherein the linker is a carbonyl or thiocarbonyl group.</u><u style="single">(Item 174)</u><u style="single">166. The kit of item 166, wherein each reagent in the above set separately binds to a solid support via a cleavable linker.</u><u style="single">(Item 175)</u><u style="single">166. The kit of item 166, wherein all reagents in the above set are isomers.</u><u style="single">(Item 176)</u><u style="single">166. The kit of item 166, wherein all reagents in the above set are isobaric.</u><u style="single">(Item 177)</u><u style="single">All reagents in the above set have the following formula:</u><chemistry num="27"><img file="JP2010190902A_D0029.tif" /></chemistry><u style="single">Including</u><u style="single"> During the ceremony</u><u style="single"> a) RG is a reactive group that is an electrophile</u><u style="single"> b) Z is O, S, NH, or NR</u>^{<u style="single">1</u>}<u style="single">And</u><u style="single"> c) Each J is the same or different, H, deuterium (D), R</u>^{<u style="single">1</u>}<u style="single">, OR</u>^{<u style="single">1</u>}<u style="single">, SR</u>^{<u style="single">1</u>}<u style="single">, NHR</u>^{<u style="single">1</u>}<u style="single">, N (R)</u>^{<u style="single">1</u>}<u style="single">)</u>_{<u style="single">2</u>}<u style="single">, Fluorine, Chlorine, Bromine, and Iodine.</u><u style="single">d) W is an atom or group located in the cyclic nitrogen in ortho, meta, or para, NH, NR.</u>^{<u style="single">1</u>}<u style="single">, NR</u>^{<u style="single">2</u>}<u style="single">, PR</u>^{<u style="single">1</u>}<u style="single">, PR</u>^{<u style="single">2</u>}<u style="single">Selected from the group consisting of, O, or S, e) each carbon of the above heterocycle is represented by the formula CJ.</u>_{<u style="single">2</u>}<u style="single">Have,</u><u style="single">f) Each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is , Separately containing bonded hydrogen, deuterium, and / or fluorine atoms,</u><u style="single">g) R</u>^{<u style="single">2</u>}<u style="single">Is a cleavable linker that cleaves an aminoalkyl, hydroxyalkyl, thioalkyl group, or the above reagent to a solid support, wherein the aminoalkyl, hydroxyalkyl, or thioalkyl group is 1 to 8 pieces. It has a carbon atom of, and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, and the carbon atoms of the alkyl and aryl groups are separately bonded hydrogen, deuterium, and / or fluorine. The kit according to item 176, which contains atoms.</u><u style="single">(Item 178)</u><u style="single">The above set includes the following four types of reagents, that is,</u><chemistry num="28"><img file="JP2010190902A_D0030.tif" /></chemistry><u style="single">177. The kit according to item 177, wherein RG is the above-mentioned reactive group, which comprises one or more of the above-mentioned reaction groups.</u><u style="single">(Item 179)</u><u style="single">The above set includes the following four types of reagents, that is,</u><chemistry num="29"><img file="JP2010190902A_D0031.tif" /></chemistry><u style="single">177. The kit according to item 177, wherein RG is the above-mentioned reactive group, which comprises one or more of the above-mentioned reaction groups.</u><u style="single">(Item 180)</u><u style="single">The above set includes the following four types of support binding reagents, that is,</u><chemistry num="30"><img file="JP2010190902A_D0032.tif" /></chemistry><u style="single">Including one or more of</u><u style="single"> During the ceremony</u><u style="single">a) RG is the above reactive group</u><u style="single">b) E is a solid support</u><u style="single">c) F is a cleavable linker attached to the solid support.</u><u style="single">d) G is an aminoalkyl, hydroxyalkyl, or thioalkyl group that is cleaveable to a cleaving linker, wherein the aminoalkyl, hydroxyalkyl, or thioalkyl group contains 1 to 8 carbon atoms. It may optionally contain a heteroatom or a substituted or unsubstituted aryl group, and the carbon atoms of the alkyl and aryl groups described above separately contain bonded hydrogen, deuterium, and / or fluorine atoms. , E) Each carbon of the above heterocycle is given by the formula CJ.</u>_{<u style="single">2</u>}<u style="single">Where each J is the same or different, H, deuterium (D), R</u>^{<u style="single">1</u>}<u style="single">, OR</u>^{<u style="single">1</u>}<u style="single">, SR</u>^{<u style="single">1</u>}<u style="single">, NHR</u>^{<u style="single">1</u>}<u style="single">, N (R)</u>^{<u style="single">1</u>}<u style="single">)</u>_{<u style="single">2</u>}<u style="single">, Fluorine, Chlorine, Bromine, and Iodine.</u><u style="single">f) Each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is 177. The kit of item 177, which comprises, separately, bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">(Item 181)</u><u style="single">All reagents in the above set have the following formula:</u><chemistry num="31"><img file="JP2010190902A_D0033.tif" /></chemistry><u style="single">Including</u><u style="single"> During the ceremony</u><u style="single"> a) RG is a reactive group that is a nucleophile or electrophile</u><u style="single"> b) Z is O, S, NH, or NR</u>^{<u style="single">1</u>}<u style="single">And</u><u style="single"> c) Each J is the same or different, H, deuterium (D), R</u>^{<u style="single">1</u>}<u style="single">, OR</u>^{<u style="single">1</u>}<u style="single">, SR</u>^{<u style="single">1</u>}<u style="single">, NHR</u>^{<u style="single">1</u>}<u style="single">, N (R)</u>^{<u style="single">1</u>}<u style="single">)</u>_{<u style="single">2</u>}<u style="single">, Fluorine, Chlorine, Bromine, and Iodine.</u><u style="single"> d) Each R</u>^{<u style="single">1</u>}<u style="single">Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is The kit of item 176, which comprises, separately, bonded hydrogen, deuterium, and / or fluorine atoms.</u><u style="single">(Item 182)</u><u style="single">The above set includes the following four types of reagents, that is,</u><chemistry num="32"><img file="JP2010190902A_D0034.tif" /></chemistry><u style="single">181 of the kit, wherein RG is the above-mentioned reactive group, which comprises one or more of the above.</u></p>

<figref num="1A">FIG. 1A shows the reaction of the analyte with two isobaric labeling reagents (eg, Compounds I and II).</figref><figref num="1B">FIG. 1B shows the fragmentation of the labeled analyte shown in FIG. 1A, which produces different mass reporter moieties (eg, compounds VII and VIII as sine ions) from the isobaric labeled analyte. Will be done.</figref><figref num="2">FIG. 2 is an enlarged plot of the mass spectrum of the labeled analyte.</figref><figref num="3">FIG. 3 is a complete mass spectrum obtained from the second mass spectrometry of the selective labeling analyte identified in the enlarged plot of FIG.</figref><figref num="4">FIG. 4 is an enlarged plot of the mass spectrum of the dominant y-ion daughter fragment ions of the analyte measured in the second mass spectrometry.</figref><figref num="5">FIG. 5 is an enlarged plot of the mass spectrum of the dominant b-ion daughter fragment ions of the analyte measured in the second mass spectrometry.</figref><figref num="6">FIG. 6 is an enlarged plot of the mass spectra of the two reporters (ie, sine ions) measured in the second mass spectrometry.</figref><figref num="7">FIG. 7 is a plot of the reporter's observation-to-prediction ratio measured by second mass spectrometry for various mixtures of labeled peptides, where each peptide in the mixture contains one of two different reporters.</figref><figref num="8">FIG. 8 shows two sets of isobaric labeling reagents using the same isotope (Compounds X-XIII) and different isotopes (Compounds XV-XVIII), thereby within the set. Reporter / linker moieties of the same total mass are achieved, although they have reporter moieties of different total mass.</figref><figref num="9A">FIG. 9A shows a synthetic route for synthesizing an isotope-labeled piperazine-labeled reagent from the original starting material. This pathway can also be used in the preparation of non-isotope-labeled piperazine reagents using non-isotope-labeled starting materials.</figref><figref num="9B">FIG. 9B shows a synthetic route for synthesizing an isotope-labeled piperazine-labeled reagent from the original starting material. This pathway can also be used in the preparation of non-isotope-labeled piperazine reagents using non-isotope-labeled starting materials.</figref><figref num="10">FIG. 10 shows a synthetic route for synthesizing isotope-labeled N-alkylpiperazine-labeled reagents and non-isotope-labeled N-alkylpiperazine-labeled reagents from the original starting materials.</figref><figref num="11">FIG. 11 shows a synthetic pathway for synthesizing isotope-labeled N-alkylpiperazine-labeled reagents and non-isotope-labeled N-alkylpiperazine-labeled reagents from the original starting materials.</figref><figref num="12">FIG. 12 shows a solid phase-based synthetic pathway for synthesizing isotope-labeled piperazine-labeled reagents and non-isotope-labeled piperazine-labeled reagents from starting materials.</figref>

(4. Description of various embodiments of the present invention) (A. Method) According to the method of the present invention, the analyte to be measured is labeled. The labeled analyte, the analyte itself, one or more fragments of the analyte and / or the fragment of the label can be measured by mass spectrometry. In some embodiments, the methods of the invention include a plurality of different samples contained in the same sample, as well as for multiplex analysis of a plurality of identical and / or different analytes contained in two or more different samples. It is used for the analysis of the analyte. Two or more types of samples can be mixed into one sample mixture. In the multiplexing analysis, a standardized sample can be used to measure from which sample of the sample mixture the analysis product is derived. Absolute and / or relative (often expressed in concentration or quantity) amounts (often expressed in concentration or quantity) of the analyte (with respect to the same analyte contained in different samples), in two or more samples combined to form a sample mixture. Can be measured. In addition, mass spectrometry of fragments of an analyte (eg, daughter fragment ions) is used to identify precursors to the analyte and / or the analyte (eg, when the precursor to the analyte is degraded).

One of the features of the described approach is the unique labeling by which multiple analytes from different samples are chemically isomers or isotopes (having equal mass) and identify the sample from which the analyte is derived. It is in the fact that it can be differentially isotope-labeled (ie, isotope-encoded) by. The differentially labeled analyte is indistinguishable on an MS mode mass spectrometer. This is because all the differentially labeled analyzes have the same (total) mass-to-charge ratio. However, when subjected to dissociation energy levels, for example via collision-induced dissociation (CID), the label can be fragmented to give rise to a unique reporter that can be decomposed by mass (mass-to-charge ratio) on a mass spectrometer. .. It is possible to correlate the relative amount of the reporter observed in the mass spectrum with the relative amount of the labeled analyte contained in the sample mixture and, connotatively, the amount of the analyte contained in the sample from which the analyte is derived. it can. Therefore, the relative intensity of the reporter (ie, sine ions) is used to determine the relative amount of one or more analytes contained in two or more samples combined to form a sample mixture. Can be measured.

From the reporter information, the absolute amount (often expressed in concentration and / or quantity) of one or more analyzes contained in two or more samples, for each analysis for which absolute quantification is desired. If the calibration criteria are incorporated into the sample mixture, they can be derived.

For example, the analyte may be a peptide resulting from the degradation of a protein that uses an enzymatic digestion reaction to process the sample. Proteolysis can be achieved by treating the sample with a proteolytic enzyme (eg, trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin, or carboxypeptidase C). A precursor to a degraded peptide by measuring the identity and amount of peptide contained in the sample mixture, and by identifying the sample from which it is derived, optionally in combination with measurements of other peptides obtained from that sample. A protein can be identified and / or quantified for the sample from which it is derived. This method is a multiplex method because it allows multiple measurements of proteins in multiple samples (ie, from a sample mixture).

In some embodiments, the invention comprises reacting each of two or more samples, each containing one or more reactive analytes, with different labeling reagents in the labeling reagent set, wherein the labeling comprises the above. It relates to a method in which each of the different labeling reagents in the chemical reagent set has the formula: RP-X-LK-Y-RG. As a result, one or more analytes of each sample are subjected to the reaction of each electrophile or nucleophile reactive group (RG) of a different labeling reagent with the nucleophile or electrophile of the analyte. Marked by the "RP-X-LK-Y-" part. This labeling process produces two or more differentially labeled samples, each containing one or more labeled analytes. The labeling reagents in the above set can be isomeric or isobaric. A reporter for each labeling reagent can be identified by the sample from which each labeled analyte is derived, which can be used to identify the sample.

RG is a reactive group and its properties have already been described. The RP is the reporter part and its properties have already been described. For each reagent in the above set, the total mass of each reporter is different. LK is the linker part and its properties have already been described. The total mass of the linker can be corrected for differences in total mass between reporters for different labeling reagents such that the integrated total mass of the reporter / linker combination is the same for each reagent in the above set. X is the bond between the atom of the reporter and the atom of the linker. Y is the bond between the linker atom and the reactive group (or after the reaction with the analyte, Y is the bond between the linker atom and the analyte atom). Bonds X and Y are fragmented at least in part of the labeled analyte when subjected to dissociation energy levels on a mass spectrometer. The characteristics of the bonds X and Y have already been described.

Once the analyte of each sample is labeled with a labeling reagent specific to that sample, two or more different labeled samples or parts thereof can be mixed to produce a sample mixture. If quantification is required, the volume and / or volume of each sample combined to produce the sample mixture can be recorded. The volume and / or volume of each sample relative to the total sample volume and / or volume of the sample mixture is expressed as the amount (often concentration and / or volume) of the identified analyte contained in each sample obtained from the analysis of the sample mixture. It can be used to measure the ratio required to measure the sample. Thus, the sample mixture can include a composite mixture, with relative quantification of the same and / or different analytes, relative quantification of the amount of analyte contained in each of two or more samples, or calibration criteria. Can also be identified and / or quantified by either absolute quantification in addition to the sample mixture.

The mixture is then subjected to spectrometry techniques. In this technique, a first mass spectrometer can be used to perform a first mass spectrometry on a sample mixture or a fraction thereof. Ions with a specific mass-to-charge ratio obtained from the first mass spectrometry can then be selected. The selected ions are then exposed to dissociation energy levels (eg, collision-induced dissociation (CID)) to induce fragmentation of the selected ions. By exposing selected ions of the labeled analyte of a particular mass-to-charge ratio to dissociation energy levels, both bond X and Y can be fragmented at least a portion of the selected ions. Both bound X and Y fragmentation can cause fragmentation of the reporter / linker moiety as well as separating the charged or ionized reporter from the analyte. Ions exposed to dissociation energy levels can also cause fragmentation of the analyte, thereby producing daughter fragments / ions of the analyte. Ions (remaining selected ions, daughter fragment ions, or charged or ionized reporters), or fractions thereof, can be directed to a second mass spectrometer.

In the second mass spectrometer, a second mass spectrometry is performed on the selected ions and their fragments. The second mass spectrometry is the total mass (or m / m /) of each unique reporter present at the selected mass-to-charge ratio, as well as the total mass of the daughter fragment ions of at least one reactive analysis of the sample mixture. z) and relative quantities can be measured. For each analyte present at a selected mass-to-charge ratio, daughter fragment ions can be used to identify one or more analytes present at a selected mass-to-charge ratio. For example, this analysis can be performed as already described in the section entitled "Analyst Measurements by Computer-Assisted Database Analysis".

In some embodiments, some steps of the process can be repeated one or more times. For example, in some embodiments, the ions of the mass-to-charge ratio selected from the analysis by the first mass spectrometry, which are different from any of the mass-to-charge ratios already selected, have been processed for the dissociation energy level and are already As described, at least some ionization daughter fragment ions and ionization reporter moieties of the selected ions are formed. A second mass spectrometry of selected ions, ionized reporter moieties, and daughter fragment ions, or fractions thereof, can be performed. It is also possible to measure the total mass and relative mass of each reporter portion and the total mass of daughter fragment ions in the second mass spectrometry. Thus, this information can be used to identify and quantify one or more other analytes from the first mass spectrometry.

In some embodiments, the entire process can be repeated one or more times. For example, if the sample mixture is fractionated (eg separated by chromatography or electrophoresis), it may be useful to repeat the process one or more times. By repeating the process for each sample, it is possible to analyze the entire sample mixture. In some embodiments, it is conceivable that the entire process is repeated one or more times, and within each of these iterations, a particular step is also repeated one or more times as described above. In this way, the content of the sample mixture can be queried and measured in the best possible range.

Those skilled in the art of mass spectrometry understand that the first and second mass spectrometry can be performed on a tandem mass spectrometer. Suitable instruments for performing tandem mass spectrometry have already been described herein. A tandem mass spectrometer is preferred, but a one-stage mass spectrometer may be used. For example, analyte fragmentation can be the result of cone voltage fragmentation, followed by mass spectrometry of the resulting fragments using a one-stage quadrupole or time-of-flight mass spectrometer. In another embodiment, a laser source can be used to expose the analyte to dissociation energy / level, and the resulting fragments are on a time-of-flight or tandem time-of-flight (TOF-TOF) mass spectrometer. Can be recorded after post-source decomposition.

Based on the multiplex method disclosed above, in some embodiments, binding X is more prone to fragmentation compared to fragmentation of the binding of the analyte (eg, the amide (peptide) binding in the peptide backbone). High, low, or substantially equal. In some embodiments, the binding Y is more or less prone to fragmentation compared to the fragmentation of the binding of the analyte (eg, the amide (peptide) bond in the peptide backbone). In some embodiments, the linker for each reagent in the above set is neutral in charge after fragmentation of bound X and Y (ie, the linker is fragmented resulting in mass neutral loss, hence the MS / MS spectrum. Not observed). In yet some other embodiments, the positions of the binding X and Y are within multiple standardized reagents in a set, in multiple labeling analyzes of a mixture, or in multiple labeling of a kit. Does not change in the reagent. In yet some other embodiments, the reporter for each reagent in the above set is substantially sub-under the conditions used to fragment the analyte (eg, the amide (peptide) bond of the peptide backbone). Do not fragment. In yet some other embodiments, the bond X is less prone to fragmentation than the bond Y. In yet some other embodiments, the bond Y is less prone to fragmentation than the bond X. In some other embodiments, the bindings X and Y have approximately the same instability, otherwise one fragmentation of the binding X or Y comprises the other fragmentation of the binding X or Y. , Will be selected. Another property of the group relative to the RP-X-LK-Y-part of the labeled analyte has already been described.

In some embodiments, the label of each isobaric labeling assay is a 5, 6 or 7-membered heterocycle containing a ring nitrogen atom, which ring nitrogen atom is N-alkyl by a substituted or unsubstituted acetic acid moiety. The analyte is attached via the carbonyl carbon of the N-alkylacetic acid moiety. Here, each different label can contain one or more heavy atom isotopes. The heterocycle may be substituted or unsubstituted. The heterocycle can be aliphatic or aromatic. Possible substituents on the heterocyclic moiety include alkyl, alkoxy, and aryl groups. Substituents can include protected or unprotected groups suitable for attaching the analyte to the support, such as amine, hydroxyl, or thiol groups. The heterocycle can further contain one or more heteroatoms such as nitrogen, oxygen, or sulfur atoms.

In some embodiments, the labeled analytes contained in the sample mixture can be isobaric, each having the following general formula: That is,

<chemistry num="35"><img file="JP2010190902A_D0035.tif" /></chemistry> In the formula, Z is O, S, NH, or NR¹; Each J is the same or different, H, deuterium (D), R¹, OR¹, SR¹, NHR¹, N (R)¹)₂, Fluorine, chlorine, bromine, or iodine; W is an atom or group located at ortho, meta, or para with respect to ring nitrogen, NH, NR¹, NR², PR¹, PR², O, or S; each carbon in the heterocycle is in equation CJ₂Have; each R¹Are the same or different, optionally heteroatoms or alkyl groups with 1 to 8 carbon atoms, including substituted or unsubstituted aryl groups, wherein the alkyl or aryl group carbon atoms are separate. Contains bonded hydrogen, deuterium, and / or fluorine atoms; in addition, R²Is a cleavable linker that cleaves an aminoalkyl, hydroxyalkyl, thioalkyl group, or reagent to a solid support, where the aminoalkyl, hydroxyalkyl, thioalkyl group is 1 to 8 carbon atoms. And optionally contain a heteroatom or a substituted or unsubstituted aryl group, and the carbon atoms of the alkyl and aryl groups are separately bonded hydrogen, deuterium, and / or. Contains fluorine atoms.

For example, the sample mixture can include one or more isobaric labeled analytes represented by the following formula.

<chemistry num="36"><img file="JP2010190902A_D0036.tif" /></chemistry>In the formula, the isotopes of carbon-13 and oxygen-18 are used to balance the total mass between the morpholine reporter and the carbonyl linker of different labeling reagents.

Morpholine-labeled reagents suitable for producing labeled analytes of this general structure can be prepared by a number of synthetic routes. For example, an isotope-labeled morpholine compound or an isotope-unlabeled morpholine compound can be combined with an isotope-labeled bromoacetic acid compound or an isotope-unlabeled bromoacetic acid compound, as described in Example 1. Can be reacted. Equally apparent, the ring-substituted morpholin and / or substituted bromoacetic acid starting materials also vary little or completely from the methods described above or other methods well known to those of skill in the art without undue experimentation. Selected and used by one of ordinary skill in the art (without adding), which can produce a variety of different labeling reagents based on morpholine, the labeling reagents having different heavy atom isotope content (ie, ie). It can be used in sets or kits of the present invention (isotopically encoded).

Instead of morpholine, it is possible to select a substituted or unsubstituted piperidine with the desired isotopic distribution. If piperidine is selected, isotope D (deuterium),¹³C, or¹⁵N is H,¹²C, and¹⁴Can be replaced with N respectively, in the case of piperidine¹⁸It can be used to alter the total mass of reagents in a set of labeling reagents in a manner similar to that described for morpholine, except that O is not used as a ring atom. Typical piperidine synthesis uses an optionally isotope-enriched starting material and is described in Example 6.

The sample mixture can include one or more isobaric labeled analytes represented by the following formula.

<chemistry num="37"><img file="JP2010190902A_D0037.tif" /></chemistry>In the formula, the isotopes of carbon-13, oxygen-18, and nitrogen-15 are used to balance the total mass between the reporters of the different labeling reagents and the carbonyl linker. Piperazine labeling reagents suitable for producing labeled analytes of this general structure can be prepared by multiple synthetic routes. For example, a heavy or light piperazine compound can be reacted with a heavy or lightly labeled bromoacetic acid compound as described in Example 7. Figures 9A and 9B show two different synthetic pathways for isotope-enriched piperazine using readily available heavy or light starting materials.

Especially referring to Figure 9A, 2 equivalents¹⁵N-labeled glycine 1 can be condensed to form bis-isotope-labeled diketopiperazine 2 (isotope-labeled with * in the figure). Next, diketopiperazine 2 is reduced to isotope-labeled piperazine. The isotope-labeled piperazine is reacted with bromoacetic acid to convert it to the active ester 3, as shown in Example 7.

Especially with reference to Figure 9B¹⁵N-labeled ethylenediamine 4 can be condensed with oxalic acid 5 to give bis-isotope-labeled diketopiperazine 6 (isotope-labeled with * in the figure). The diketopiperazine can then be reduced to isotope-labeled piperazine. In addition, the isotope-labeled piperazine is reacted with bromoacetic acid to convert it to the active ester 3, as shown in Example 7.

Equally clear, ring-substituted piperazine can be made using the above method by simply selecting an appropriately substituted starting material. Where appropriate, substituted bromoacetic acid (either heavy or light) can be used as well. Heavy means that a compound is isotope-enriched by one or more heavy atom isotopes. Light means that it is not isotopically enriched.

Therefore, by selecting an appropriately substituted starting material, various different piperazine-based labeling reagents that can be used in the set of the present invention can be produced.

For example, the sample mixture can include one or more isobaric labeled analytes represented by the following formula. That is,

<chemistry num="38"><img file="JP2010190902A_D0038.tif" /></chemistry>In the formula, the isotopes of carbon-13, oxygen-18, and nitrogen-15 are used to balance the total mass between the reporters of the different labeling reagents and the carbonyl linker. Here, (1) each R¹Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is. Separately, it contains bonded hydrogen, deuterium, and / or fluorine atoms; and (2) each K is separately selected as a hydrogen or amino acid side chain. Substituted piperazine labeling reagents suitable for producing labeled analytes of this general structure can be prepared by a number of synthetic routes.

For example, with reference to FIG. 10, the N-alkyl substituted piperazine reagent can be synthesized according to the procedure shown. Esters of t-boc-protected glycine-N-methyl-glycine dimer 12 by condensing t-butoxycarbonyl (t-boc) -protected glycine 10 with an ester of N-methyl-glycine 11 (eg, an ethyl ester) Can be formed. The t-boc protecting group can then be removed to cyclize the glycine dimer 12 followed by condensation to form the salt of N-methyl-diketopiperazine 13. N-methyl-piperazine 14 is formed by neutralizing and reducing 13 acid salts. N-Methyl-piperazin 14 can then be reacted with bromoacetic acid 15 (or a substituted version thereof) to convert it to the active ester 16 as shown in Example 7.

What is clear is that the ring-substituted piperazine is simply selected from amino acids other than glycine or N-methyl amino acids (or esters thereof) (eg, alanine, phenylalanine, leucine, isoleucine, valine, asparagine, aspartic acid, etc.). , It means that it can be made by using the above method. Also apparent is that the amino acids are isotope-labeled in a manner suitable for the preparation of ring-substituted piperazines with the desired distribution of isotopes required to prepare a set of isotopic labeling reagents. Is possible.

The N-alkyl substituted piperazine reagent can be prepared by yet another illustrated procedure. With reference to FIG. 11, the glycine methyl ester 21 can be reacted with the ethyl ester of bromoacetic acid 22 to form diethyliminodiacetate 23. The diester of diethyliminodiacetate 23 can be converted to the dioxide 24 by treatment with a suitable reagent (eg, thionyl chloride). The dichloride 24 can then be reacted, for example, with an alkylamine (eg, methylamine) to form the N-alkyl-diketopiperazine 25. The N-alkyl-di-ketopiperazine 25 can then be reduced to form the N-alkyl-piperazine 26. The N-alkyl-piperazine can then be reacted with bromoacetic acid to convert it to the active ester 27, as shown in Example 7.

It is clear that the ring-substituted piperazine is simply selected as an ester of an amino acid other than glycine (eg, alanine, phenylalanine, leucine, isoleucine, valine, asparagine, aspartic acid, etc.) or a substituted version of bromoacetic acid. It means that it can be made using the method. Equally obvious, amino acids and bromoacetic acid (and its substituted derivatives) are suitable for the preparation of ring-substituted piperazines with the desired distribution of isotopes required to prepare a set of isotopic labeling reagents. It means that it can be isotope-labeled in the same way. More clearly, the selection of alkyldiamines, hydroxylalkylamines, or thioalkylamines, or isotope-labeled versions thereof, in place of alkylamines is described in more detail below. It means that it can be used to generate a support binding labeling reagent.

In yet another embodiment of the method, the labeled analyte of the sample mixture is isobaric, each composed of the following equations: That is,

<chemistry num="39"><img file="JP2010190902A_D0039.tif" /></chemistry>In the formula, Z is O, S, NH, or NR¹; Each J is the same or different, H, deuterium (D), R¹, OR¹, SR¹, NHR¹, N (R)¹)₂, Fluorine, Chlorine, Bromine, and Iodine;¹Are the same or different, optionally heteroatoms or alkyl groups with 1 to 8 carbon atoms, including substituted or unsubstituted aryl groups, wherein the alkyl or aryl group carbon atoms are separate. Includes, bonded hydrogen, deuterium, and / or fluorine atoms.

For example, the sample mixture can include two or more isobaric labeled analytes represented by the following formula. That is,

<chemistry num="40"><img file="JP2010190902A_D0040.tif" /></chemistry>In the formula, carbon-13 and oxygen-18 isotopes are used to balance the total mass between the reporters of the different labeling reagents and the carbonyl linker. Substitution labeling reagents suitable for producing labeled analytes of this general structure can be prepared by the general process described in Example 8.

In yet another embodiment of the invention, each different labeling reagent constituting a set or kit of labeling reagents allows each different sample to react with a support carrying a different labeling reagent. Can be attached to the support via a cleavable linker. In some embodiments, the support is itself used to label the reactive analyte. In some embodiments, the labeling reagent can be removed from the support and, in some cases, after subsequent processing (eg, protection of reactive groups), can be used to label the reactive analyte.

Based on some embodiments, the sample analyte can react with a solid support (each sample reacts with a different solid support and thus with a different reporter) and does not react with reactive groups. The resin binding component of the sample can be arbitrarily washed away. One or more labeled analytes are then removed from each solid support by treating the support under conditions where the cleavable linker is cleaved, thereby removing from the support. The reporter / linker / analyte complex can be released. Each support contains one or more labeled analytes, as each support can be treated similarly under conditions where two or more different samples can be obtained by cleaving the cleaveable linker. .. Here, the labeled analyte associated with a particular sample is identified and / or quantified by a unique reporter associated with it. Next, as already mentioned, the recovered samples can be mixed to form a sample mixture.

For example, each different labeling reagent in the set used in the method described above can be a solid support of formula: EF-RP-X-LK-Y-RG. In the formula, RG, X, Y, RP, and LK have already been described. E is a solid support and F is a cleavable linker that is cleavably attached to the reporter and is attached to the solid support. The support of this general formula can be prepared as described in Example 9.

In some embodiments, the set of support binding labeling reagents is based on a labeled N- (aminoalkyl) piperazine derivative, an N- (thioalkyl) piperazine derivative, or an N- (hydroxyalkyl) -piperazine derivative. Can be done. Both heavy and light piperazine derivatives can be prepared. The labeled N- (aminoalkyl) piperazine derivative, N- (thioalkyl) piperazine derivative, or N- (hydroxyalkyl) -piperazine derivative is initiated, for example, with an alkyldiamine, thioalkylamine, or hydroxyalkylamine as the N-alkylamine. It can be formed using the procedure shown in FIG. 11 (see discussion in FIG. 11 above). When alkyldiamines, thioalkylamines, or hydroxyalkylamines are suitable for the synthesis of the desired N- (aminoalkyl) piperazine derivative, N- (thioalkyl) piperazine derivative, or N- (hydroxyalkyl) -piperazine derivative. , Heavy or light. The amino, hydroxyl, or thiol groups of the N- (aminoalkyl) piperazine derivative, the N- (thioalkyl) piperazine derivative, or the N- (hydroxyalkyl) -piperazine derivative can be protected, if desired. When using alkyldiamines, thioalkylamines, or hydroxyalkylamines, the piperazine can include N-aminoalkyl, N-thioalkyl, or N-hydroxyalkyl moieties. Here, by reacting the amino, hydroxyl, or thiol group of this portion with a cleaving linker on the support, an N- (aminoalkyl) piperazine derivative, an N- (thioalkyl) piperazine derivative, or N- (hydroxy) Alkyl) -Piperazine prepared from a piperazine derivative can be attached to the support in a cleavable manner.

A support containing a labeling reagent can be prepared by any of several methods. In some embodiments, the amino, hydroxyl, or thiol groups of N- (aminoalkyl) piperazine, N- (thioalkyl) piperazine, or N- (hydroxyalkyl) -piperazine are cleaved in a suitable support. Can be reacted with. The cleavable linker is "sterically hindered cleavable. Linker) (see Example 9). Piperazin can be reacted with isotope-labeled or non-isotope-labeled haloacetic acids (substitution or unsubstituted), depending on the nature of the labeling reagent required for the set of labeling reagents. The carboxylic acid can then be converted to an active ester. The active ester can react with the analyte of the sample and label the analyte with a support labeling reagent. When the cleavable linker is cleaved, the labeled analyte is released from the support. This process is repeated with a unique piperazine-based labeling reagent to prepare the different supports that make up the set of labeled supports.

In some embodiments, N- (aminoalkyl) piperazine, N- (thioalkyl) piperazine, or N- (hydroxyalkyl) -piperazine is an isotope-labeled or non-isotope-labeled haloacetic acid (substituted or unsubstituted). ), Or its ester can be reacted first. Preferably, the amino, hydroxyl, or thiol groups of N- (aminoalkyl) piperazine, N- (thioalkyl) piperazine, or N- (hydroxyalkyl) -piperazin can be protected with a suitable protecting reagent (suitable). For a list of protecting groups, see Green et al., Protecting Groups In Organic Synthesis, Third Edition, John Wiley &amp; Sons, Inc., New York, See 1999). The unprotected amino, thiol, or hydroxyl group of the resulting bis-alkylated piperazine can then react with a cleavable linker on a suitable support. The carboxylic acid is then converted to an active ester. If the haloacetic acid compound is an ester, the ester can be saponified prior to conversion to an active ester. The active ester can be reacted with the sample analyte and labeled with the support labeling reagent. When the cleavable linker is cleaved, the labeled analyte is released from the support. This process is repeated with a unique piperazine-based labeling reagent to prepare the different supports that make up the set of labeled supports.

Thus, in some embodiments, the set of labeling reagents can include one or more of the following support binding labeling reagents: That is,

<chemistry num="41"><img file="JP2010190902A_D0041.tif" /></chemistry> In the formula, RG, E, and F have already been described. According to the method, G is an aminoalkyl, hydroxyalkyl, or thioalkyl group that is cleaveable to a cleavable linker, where the aminoalkyl, hydroxyalkyl, or thioalkyl group is 1 It may contain up to 8 carbon atoms and optionally a heteroatom or a substituted or unsubstituted aryl group, wherein the alkyl and aryl group carbon atoms are separately bonded hydrogen, deuterium, and the like. And / or contains fluorine atoms. Each carbon in the heterocycle has the formula CJ₂And each J is the same or different, H, deuterium (D), R¹, OR¹, SR¹, NHR¹, N (R)¹)₂, Fluorine, chlorine, bromine, or iodine, each R¹Are the same or different, optionally heteroatoms or alkyl groups with 1 to 8 carbon atoms, including substituted or unsubstituted aryl groups, wherein the alkyl or aryl group carbon atoms are separate. Includes, bonded hydrogen, deuterium, and / or fluorine atoms.

In some embodiments, the labeled analyte is prepared by first reacting the analyte with a support containing a labeling reagent that is cleavably attached to the support via a cleavable linker. It can be produced by cutting from the support. Therefore, the sample mixture can contain one or more isobaric labeled analytes represented by the following formula. That is,

<chemistry num="42"><img file="JP2010190902A_D0042.tif" /></chemistry>In the formula, G'is an aminoalkyl, hydroxyalkyl, or thioalkyl group having 1 to 8 carbon atoms, optionally containing heteroatoms or substituted or unsubstituted aryl groups, wherein the alkyl and aryl groups. Carbon atoms separately contain bonded hydrogen and / or deuterium. Each carbon of the heterocycle is given by the formula CJ₂Where each J is the same or different, H, deuterium (D), R¹, OR¹, SR¹, NHR¹, N (R)¹)₂, Fluorine, chlorine, bromine, and iodine. Each R¹Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is. Separately, it contains bonded hydrogen, deuterium, and / or fluorine atoms. Here, an alkylamine group, a hydroxyalkyl group, or a thioalkyl group can be a moiety bonded to a cleavable linker of a solid support. The products of each cleavage reaction can be combined to produce a sample mixture suitable for analysis of labeled analytes by the methods described herein.

In some embodiments, the method of the invention is the step of digesting each sample with at least one enzyme and partially or completely degrading the components of the sample prior to labeling the analyte of the sample. Can be further included (see also the section above entitled "Sample Processing"). For example, the enzyme can be a protease (to degrade proteins and peptides) or a nuclease (to degrade nucleic acids). Moreover, you may decompose a sample component by using a plurality of enzymes together. The enzyme can be a proteolytic enzyme such as trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin, or carboxypeptidase C.

In some embodiments, the method can further include separating the sample mixture prior to performing the first mass spectrometry (see also section above entitled "Separation of Sample Mixture"). thing). In this way, the first mass spectrometry is performed only on the fraction of the sample mixture. The above separation can be carried out by any separation method, and the separation method includes chromatography or electrophoresis. For example, liquid chromatography / mass spectrometry (LC / MS) can be used to perform such sample separation and mass spectrometry. In addition, any chromatographic separation process suitable for separating the analyte of interest can be used. A non-limiting example of a suitable chromatographic and electrophoretic separation process is described herein.

In yet another embodiment, the method of the invention can include both an enzymatic treatment and a separation step of degrading the sample constituents.

As described above, by analyzing the total mass of daughter fragment ions, it is possible to measure the analyte associated with the selected ions. One such measurement method is described in the section entitled "Measurement of Analysts by Computer-Assisted Database Analysis". Once the analyte is measured, the information about the total mass and relative mass of each reporter portion in the second mass spectrometry and the total mass of the daughter fragment ions provides the basis for determining other information about the sample mixture. To do. The amount of reporter can be measured by the peak intensity in the mass spectrum. In some embodiments, the amount of reporter can be measured by analyzing the peak height or peak width of the reporter (sine ion) signal obtained using a mass spectrometer. Measurement of different reporters in the second mass analysis because each sample can be labeled with a different labeling reagent and each labeling reagent can contain a unique reporter that can correlate with a particular sample. Identify the sample from which the ions of the selected analyte are derived. If a large number of reporters are found (eg, based on the multiplexing method of the invention), the relative amount of each reporter with respect to other reporters can be determined. The relative amount of each reporter measured in the second mass spectrometry correlates with the relative amount of the analyte contained in the sample mixture, so the relative amount of the analyte in each sample combined to form the sample mixture. Can be measured (often expressed in concentration and / or quantity). If desired, reporter-associated peak intensity corrections can be made for naturally occurring or artificially generated isotopic abundance, as described above. More specifically, if the volume and / or quantity of each sample bound to the sample mixture is known, the relative amount of each sample (often expressed as concentration and / or quantity) is referred to as the second mass. It can be calculated based on the relative amount of each reporter measured in the analysis. This analysis shows selected ions with different mass-to-charge ratios. It is repeated one or more times with respect to, whereby the relative amount of one or more different analytes contained in each sample combined to form a sample mixture can be obtained. If necessary, reporter-associated peak intensity corrections are performed for naturally occurring or artificially generated isotope abundance.

Alternatively, if a calibration criterion containing a unique reporter bound to the analyte with the selected mass-charge ratio is added to the sample mixture in known amounts (often expressed as concentration / or quantity), the calibration criteria are associated. The amount of unique reporter can be used to determine the absolute amount (often expressed as concentration and / or quantity) of the analyte of each sample that combines to form a sample mixture . This is possible. Because the amount of analyte associated with the reporter for calibration criteria is known, and all relative quantities of other reporters are measured against the labeled analyte associated with the selected ion. Because it can be done. Because the relative amount of reporter measured for each of the unique reporters (including the reporter for calibration criteria) is proportional to the amount of analyte associated with each sample that combines to form a sample mixture. , The absolute amount of analyte contained in each sample (often expressed as concentration and / or quantity) shall be measured according to the calculated ratio to the formulation used to produce the sample mixture. Can be done. If necessary, reporter-associated peak intensity corrections are performed for naturally occurring or artificially generated isotope abundance.

This analysis is repeated one or more times for selected ions with different mass-to-charge ratios to obtain the absolute amount of one or more different analytes contained in each sample that combine to form a sample mixture. be able to. If necessary, reporter-associated peak intensity corrections are performed for naturally occurring or artificially generated isotope abundance.

In some embodiments, the method can be performed with a digestion and / or separation step. In some embodiments, the steps of the method with or without digestion and / or separation steps are repeated one or more times to include one or more other reagents or two or more samples contained in one sample. One or more analytes contained in (including samples labeled with support binding labeling reagents) can be identified and / or quantified. Depending on whether the calibration criteria are present in the sample mixture of a particular analyte, the quantification can be relative to other labeled analytes, or it can be absolute. Such analytical methods are particularly useful for proteomix analysis of multiple samples of one complex property, especially for preliminary separation of labeled analytes (eg, liquid chromatography or electrophoretic separation) as first mass spectrometry. This is the case prior to.

In some embodiments, the peptides contained in the sample or sample mixture can be the analyte. Analysis of peptides contained in a sample or sample mixture is used to measure the amount of identifiable protein contained in a sample or sample mixture (often expressed as concentration and / or quantity) and is used to measure one or more samples. The proteins contained in are degraded prior to the first mass spectrometry. In addition, information from different samples is compared for measurement purposes, for example to compare effects on the amount of intracellular protein cultured at varying concentrations of substances that may affect cell proliferation. be able to. Other non-limiting examples include comparison of expressed protein components in affected or healthy tissues or cell cultures. This can include comparison of expressed protein levels of cells, tissues, or biological fluids after infection by pathogens such as bacteria or viruses or other disease states (eg, cancer). In another example, it is possible to study the changes in protein concentration over time (changes over time) to determine the effect of drug therapy on the expressed protein components of cells or tissues. In yet another example, using information obtained from different samples taken over time, the concentration of a particular protein in a tissue, organ, or biological fluid as a result of a disease (eg, cancer) or infection. Can be detected and monitored.

In some embodiments, the analyte is a nucleic acid fragment contained in a sample or sample mixture. The information on the nucleic acid fragment is used to determine the amount of identifiable nucleic acid molecules (often expressed as concentration and / or quantity) contained in the sample or sample mixture, and the sample is subjected to first mass spectrometry. Disassembled prior to. Furthermore, the information obtained from different samples can be compared for the purpose of making measurements as described above.

(B. Mixture) In some embodiments, the invention relates to a mixture (ie, a sample mixture). The mixture can contain at least two differentially labeled analytes, each of which can result from a different sample and also has the formula RP-X-LK-Y-analyte. .. For each different label, some of the labeled analytes of the mixture are the same and some of the labeled analytes are different. Atoms, partials or bonds, X, Y, RP, and LK have already been described and their properties have been disclosed. Mixtures are formed by mixing all or part of the products of two or more labeling reactions. Here, in each labeling reaction, different labeling reagents of the general formula RP-X-LK-Y-RG are used, and in the formula, atoms, portions, or bonds X, Y, RP, LK, RG have already been used. It has been described and their properties have been disclosed. The labeling reagent is an isotopic-encoded isomer or isobaric labeling reagent. A unique reporter for each different labeling reagent can indicate which labeling reaction each of the two or more labeled assays came from. Labeling reagents are isomeric or isobaric. Therefore, two or more labeled analyzes of the mixture are isomeric or isobaric. As disclosed in any of the above methods, the mixture is a sample mixture. The properties of labeling reagents and labeled analytes associated with these methods have already been described.

The analysis of the mixture can be a peptide. The analysis of the mixture can be a protein. The analysis of the mixture can be peptides and proteins. The analysis product of the mixture can be a nucleic acid molecule. The analysis of the mixture can be a carbohydrate. The analysis of the mixture can be a lipid. The analysis of the mixture can be a steroid. The analysis of the mixture can be small molecule less than 1,500 daltons. The analysis product of the mixture contains two or more types of analysis products. Species to be analyzed are selected from, for example, peptides, proteins, nucleic acids, carbohydrates, lipids, steroids, and / or small molecules less than 1,500 daltons.

In some embodiments, the label of each isotopic labeled analyte is N-alkylated by a substituted or unsubstituted acetate moiety to which the analyte is attached via the carbonyl carbon of the N-alkylacetic acid moiety. A 5, 6, or 7-membered heterocycle with, each with a different label containing one or more heavy atom isotopes. The heterocycle can be substituted or unsubstituted. The heterocycle can be aliphatic or aromatic. Possible substituents on the heterocyclic moiety include alkyl, alkoxy, and aryl groups. Substituents can include protected or unprotected groups suitable for attaching the analyte to the support, such as amine, hydroxy, or thiol groups. The heterocycle can further comprise one or more heteroatoms such as nitrogen, oxygen, or sulfur atoms.

In some embodiments, the labeled analyte of the mixture is isobaric and each has the following formula: That is,

<chemistry num="43"><img file="JP2010190902A_D0043.tif" /></chemistry>In the formula, Z, J, and W have already been described and their properties are disclosed. For example, the sample mixture contains one or more isobaric labeling analytes represented by the following formula. That is,

<chemistry num="44"><img file="JP2010190902A_D0044.tif" /></chemistry>In the formula, the isotopes of carbon-13 and oxygen-18 are used to balance the total mass between the morpholine reporter and the carbonyl linker of different labeling reagents.

In some embodiments, the sample mixture can include one or more isobaric labeled analytes represented by the following formulas. That is,

<chemistry num="45"><img file="JP2010190902A_D0045.tif" /></chemistry>In the formula, the isotopes of carbon-13, oxygen-18, and nitrogen-15 are used to balance the total mass between the reporters of the different labeling reagents and the carbonyl linker. In some embodiments, the sample mixture can include one or more isobaric labeled analytes represented by the following formulas. That is,

<chemistry num="46"><img file="JP2010190902A_D0046.tif" /></chemistry>In the formula, the isotopes of carbon-13, oxygen-18, and nitrogen-15 are used to balance the total mass between the reporters of the different labeling reagents and the carbonyl linker, and in the formula, (1) each R¹Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is , Separately containing bonded hydrogen, deuterium, and / or fluorine atoms, and (2) each K is separately a hydrogen or amino acid side chain.

In some embodiments, the labeled analytes of the mixture are isobaric, each having the following formula: That is,

<chemistry num="47"><img file="JP2010190902A_D0047.tif" /></chemistry>In the formula, Z, J, and R¹Have already been described and their properties are also disclosed. For example, the sample mixture can include one or more isobaric labeled analytes represented by the following formula. That is,

<chemistry num="48"><img file="JP2010190902A_D0048.tif" /></chemistry>In the formula, carbon-13 and oxygen-18 isotopes are used to balance the total mass between the reporters of the different labeling reagents and the carbonyl linker.

In another embodiment, the labeled analyte supports the labeled analyte by first reacting the analyte with a support containing a labeling reagent that is cleavably attached to the support via a cleavable linker. It can be produced by cutting from the body. For example, a labeled analyte of a mixture is one or more isobaric with the following general formula: That is,

<chemistry num="49"><img file="JP2010190902A_D0049.tif" /></chemistry>In the formula, G'has already been described and its properties are also disclosed.

(C. Kit) In some embodiments, the present invention relates to a kit. The kit can include a set of two or more labeling reagents of the formula: RP-X-LK-Y-RG and one or more reagents, containers, enzymes, buffers, and / or instructions. Atoms, parts, bonds X, Y, RP, LK, RG have already been described and their properties are also disclosed. The labeling reagents of the kit can be isomeric or isobaric. Other properties of the kit's standardized reagents are disclosed as well. For example, the kit is useful for multiplex analysis of one or more analytes contained in the same sample or two or more different samples.

In some embodiments, the label of each isotopic labeled analyte is N-alkylated by a substituted or unsubstituted acetate moiety to which the analyte is attached via the carbonyl carbon of the N-alkylacetic acid moiety. A 5, 6, or 7-membered heterocycle with, each with a different label containing one or more heavy atom isotopes. The heterocycle can be substituted or unsubstituted. The heterocycle can be aliphatic or aromatic. Possible substituents on the heterocyclic moiety include alkyl, alkoxy, and aryl groups. Substituents can include protected or unprotected groups suitable for attaching the analyte to the support, such as amine, hydroxy, or thiol groups. The heterocycle can further comprise one or more heteroatoms such as nitrogen, oxygen, or sulfur atoms.

In some embodiments, the different reagents in the kit are isobaric, each having the following formula: That is,

<chemistry num="50"><img file="JP2010190902A_D0050.tif" /></chemistry>In the formula, RG, Z, J, and W have already been described and their properties are also disclosed. For example, the kit's reagents can include one or more isobaric labeling analytes represented by the following formula. That is,

<chemistry num="51"><img file="JP2010190902A_D0051.tif" /></chemistry>In the formula, RG is the reactive group and the isotopes of carbon-13 and oxygen-18 are used to balance the total mass between the morpholine reporter and the carbonyl linker of the different labeling reagents.

In some embodiments, the kit can include one or more isobaric labeling reagents represented by the following formulas. That is,

<chemistry num="52"><img file="JP2010190902A_D0052.tif" /></chemistry>In the formula, RG is the reactive group and the isotopes of carbon-13, oxygen-18, and nitrogen-15 are used to balance the total mass between the reporters of the different labeling reagents and the carbonyl linker.

In some embodiments, the kit's reagents can include one or more isobaric labeling reagents represented by the following formulas. That is,

<chemistry num="53"><img file="JP2010190902A_D0053.tif" /></chemistry>In the formula, carbon-13, oxygen-18, and nitrogen-15 isotopes are used to balance the total mass between the reporters of the different labeling reagents and the carbonyl linker, and in the formula, (1) each R.¹Is an alkyl group having 1 to 8 carbon atoms which is the same or different and may optionally contain a heteroatom or a substituted or unsubstituted aryl group, wherein the carbon atom of the alkyl or aryl group is , Separately containing bonded hydrogen, deuterium, and / or fluorine atoms, and (2) each K is separately a hydrogen or amino acid side chain. In yet another embodiment, the labeled analytes of the kit are isobaric, each having the following formula: That is,

<chemistry num="54"><img file="JP2010190902A_D0054.tif" /></chemistry>In the formula, RG, Z, J, and R¹Have already been described and their properties are also disclosed. For example, the kit's reagents can include one or more isobaric labeling analytes represented by the following formula. That is,

<chemistry num="55"><img file="JP2010190902A_D0055.tif" /></chemistry>In the formula, RG is the reactive group and the isotopes of carbon-13 and oxygen-18 are used to balance the total mass between the reporter of the different labeling reagents and the carbonyl linker.

In some embodiments, the present invention relates to a plurality of kits comprising one or more sets of supports, each support being cleavably attached to the support via a cleavable linker for different labeling. Contains reagents. For example, a cleavable linker is chemically or photodegradable. The support can react with different samples, whereby the sample analytes can be labeled with the same reporter / linker, and the analytes of different samples can be labeled with different reporter / linker combinations. .. A set of supports that can be used in embodiments of the present invention has the general formula: EFG-RP-X-LK-Y-RG, in which E, F, G, RP, X, LK, Y, And RG have already been described and their properties are also disclosed. Each different support in the above set can include a unique reporter.

For example, the support of the kit can include two or more kinds of reagent supports represented by the following formulas. That is,

<chemistry num="56"><img file="JP2010190902A_D0056.tif" /></chemistry>In the formula, E, F, G, and RG have already been described and their properties are also disclosed.

In some embodiments, the kit comprises a proteolytic enzyme. The proteolytic enzyme can be trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin, or carboxypeptidase C. In some embodiments, the kit can include instructions for differentially labeling the analytes of different samples with labeling reagents.

(D. Composition) In a plurality of embodiments, the present invention relates to compositions that can be used as labeling reagents. The composition can be a labeling reagent of formula RP-X-LK-Y-RG, in which atoms, moieties or bonds X, Y, RP, LK, RG have been described above and their properties. Disclosed. The labeling reagent can be isomeric or isobaric. Other properties of the labeling reagent were disclosed as well. For example, labeling reagents are useful for multiplex analysis of one or more analytes in the same sample or in two or more different samples.

Labeling reagents can be isotopically enriched (encoded) with at least one heavy atom isotope. Labeling reagents are isotopic enriched and can contain two or more heavy atom isotopes. Labeling reagents are isotopic enriched and can contain three or more heavy atom isotopes. Labeling reagents are isotopic enriched and can contain 4 or more heavy atom isotopes. In multiple embodiments, at least one heavy atom isotope is incorporated into the carbonyl or thiocarbonyl group of the labeling reagent and at least one other heavy atom isotope is incorporated into the reporter group of the labeling reagent.

Each of the heavy atom isotopes incorporated can be present with an isotope purity of at least 80%. Each of the heavy atom isotopes incorporated can be present with an isotope purity of at least 93%. Each of the heavy atom isotopes incorporated can be present with an isotope purity of at least 96%.

The labeling reagent contains a fixed charge or contains a reporter group that can be ionized. Therefore, the reporter group can contain a basic or acidic moiety that is easily ionized. In multiple embodiments, the reporter can be a morpholine, piperidine, or piperazine compound. In multiple embodiments, the reporter can be a carboxylic acid, a sulfonic acid, or a phosphate group-containing compound. Thus, in multiple embodiments, labeling reagents can be isolated in their salt form. For example, a piperazine-containing labeling reagent can be obtained as a mono-TFA salt, a mono HCl salt, a bis TFA salt, or a bis HCl salt. The number of counterions present in the labeling reagent can depend on the number of acidic and / or basic groups present in the labeling reagent.

In multiple embodiments, the labeling reagent can include a carbonyl or thiocarbonyl linker. Labeling reagents containing a carbonyl or thiocarbonyl linker can be used in active ester form for labeling the analyte. In active esters, the alcohol group forms a leaving group (LG). In a plurality of embodiments, the active ester alcohol (LG) is of the formula.

<chemistry num="57"><img file="JP2010190902A_D0057.tif" /></chemistry>Can have, in which X is O or S. The active ester can be an N-hydroxysuccinimidyl ester.

In a plurality of embodiments, the active ester compound has a ring nitrogen atom that is N-alkylated by a substituted or unsubstituted acetate moiety to which the alcohol moiety of the active ester is attached via the carbonyl carbon of the N-alkyl acetate moiety 5, It can be a 6- or 7-membered heterocycle, where the compound is isotically enriched with one or more heavy atom isotopes. The heterocycle of the active ester can be substituted with one or more substituents. The one or more substituents can be alkyl, alkoxy, or aryl groups. The one or more substituents can be alkylamines, alkylhydroxys, or alkylthio groups. The one or more substituents can be a protected or unprotected amine group, hydroxy group, or thiol group. Heterocycles can be aliphatic. The heterocycle can be aromatic. The heterocycle can contain one or more additional nitrogen, oxygen, or sulfur atoms.

In a plurality of embodiments, the active ester compound is of formula

<chemistry num="58"><img file="JP2010190902A_D0058.tif" /></chemistry>Can be an N-substituted morpholine acetate active ester compound or a salt thereof, in which LG is the leaving group of the active ester, X is O or S, and each Z is hydrogen, deuterium separately. , Fluorine, chlorine, bromine, iodine, amino acid side chains, or optionally substituted or unsubstituted aryl groups, which are linear or branched C1 to C6 alkyl groups, wherein the alkyl or aryl group is a carbon. Each atom separately comprises a bonded hydrogen, dehydrogen, or fluorine atom. In multiple embodiments, Z can be hydrogen, deuterium, fluorine, chlorine, bromine, or iodine, separately. In multiple embodiments, Z can be hydrogen, methyl, or methoxy, separately. In a plurality of embodiments, X is¹⁶O or¹⁸It is O. The nitrogen atom in the morpholine ring¹⁴N or¹⁵Can be N. In multiple embodiments, the active ester is the formula

<chemistry num="59"><img file="JP2010190902A_D0059.tif" /></chemistry>Is a compound containing, and each C in the formula^*Separately,¹²C or¹³C, LG is the leaving group of the active ester, X is O or S, and each Z is a separate hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino acid side chain, or A linear or branched C1 to C6 alkyl group which may optionally contain a substituted or unsubstituted aryl group, wherein the carbon atoms of the alkyl or aryl group are separately bonded hydrogen, deuterium, etc. Or it contains a fluorine atom.

In a plurality of embodiments, the active ester compound is of formula

<chemistry num="60"><img file="JP2010190902A_D0060.tif" /></chemistry>Can be an N-substituted piperidine acetate active ester compound or a salt thereof, in which LG is the leaving group of the active ester, X is O or S, and each Z is hydrogen, dehydrogen, separately. , Fluorine, chlorine, bromine, iodine, amino acid side chains, or optionally substituted or unsubstituted aryl groups, which are linear or branched C1 to C6 alkyl groups, wherein the alkyl or aryl group is a carbon. Each atom separately comprises a bonded hydrogen, dehydrogen, or fluorine atom. In multiple embodiments, Z can be hydrogen, deuterium, fluorine, chlorine, bromine, or iodine, separately. In multiple embodiments, Z can be hydrogen, methyl, or methoxy, separately. In a plurality of embodiments, X is¹⁶O or¹⁸It is O. The nitrogen atom in the ring of piperidine is¹⁴N or¹⁵Can be N. In multiple embodiments, the active ester is the formula

<chemistry num="61"><img file="JP2010190902A_D0061.tif" /></chemistry>Is a compound containing, and each C in the formula^*Separately,¹²C or¹³C, LG is the leaving group of the active ester, X is O or S, and each Z is a separate hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino acid side chain, or A linear or branched C1 to C6 alkyl group which may optionally contain a substituted or unsubstituted aryl group, wherein the carbon atoms of the alkyl or aryl group are separately bonded hydrogen, deuterium, etc. Or it contains a fluorine atom.

In a plurality of embodiments, the active ester compound is of formula

<chemistry num="62"><img file="JP2010190902A_D0062.tif" /></chemistry>N-substituted piperidine acetate active ester compound or a salt thereof, in which LG is the leaving group of the active ester, X is O or S, Pg is the amine protective group, and each Z Are linear or branched C1 to C6 alkyl groups that may separately contain hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino acid side chains, or optionally substituted or unsubstituted aryl groups. Here, the carbon atom of the alkyl or aryl group separately contains a bonded hydrogen, deuterium, or fluorine atom. In multiple embodiments, Z can be hydrogen, deuterium, fluorine, chlorine, bromine, or iodine, separately. In multiple embodiments, Z can be hydrogen, methyl, or methoxy, separately. In a plurality of embodiments, X is¹⁶O or¹⁸It is O. The nitrogen atom in the piperazine ring is¹⁴N or¹⁵Can be N. In multiple embodiments, the active ester is the formula

<chemistry num="63"><img file="JP2010190902A_D0063.tif" /></chemistry>Is a compound containing, and each C in the formula^*Separately,¹²C or¹³C, LG is the leaving group of the active ester, X is O or S, Pg is the amine protective group, and each Z is a separate hydrogen, dehydrogen, fluorine, chlorine, A linear or branched C1 to C6 alkyl group that may contain a bromine, iodine, amino acid side chain, or optionally a substituted or unsubstituted aryl group, where the carbon atoms of the alkyl or aryl group are separate. Includes bonded hydrogen, heavy hydrogen, or fluorine atoms.

Although embodiments of the present invention have been described, it will be apparent to those skilled in the art that other embodiments embracing this concept can be used. Therefore, it is believed that these embodiments should not be limited to the disclosed embodiments, but rather only to the spirit and scope of the invention.

<p> The present invention will be described with reference to the following examples, but the present invention is not limited in any way.</p><p> (Example 1 Synthesis of morpholine acetic acid) Bromoacetic acid (2 g, 14.4 mole) was dissolved in tetrahydrofuran (50 mL) and added dropwise to a stirred solution of morpholine (3.76 g, 43.2 mole) in tetrahydrofuran (THF, 20 mL). The solution was stirred at room temperature for 3 days. The white solid (4.17 g) was filtered, washed with THF (100 mL) and recrystallized from warm ethanol (EtOH) (yield 2.59 g, IR 1740 cm-1). For two different homoheavy forms of morpholine acetic acid, 1-¹³C Bromoacetic acid (bromoacetic-1-¹³C acid) (Aldrich PN 27,933-1) or 2-¹³C Bromoacetic acid (bromoacetic-2-¹³Any of C acid) (Aldrich PN 27,935-8) was used in place of bromoacetic acid.</p><p> (Example 2 Synthesis of N-hydroxysuccinimide ester of morpholine acetic acid) Dimethylformamide (dried, 1.75 g, 0.024 M) was dissolved in tetrahydrofuran (dried, 30 mL). This solution was added dropwise to a stirred solution of thionyl chloride (2.85 g, 0.024 M) dissolved in tetrahydrofuran (dry, 20 mL) and cooled in an ice bath. After completion of the addition and 30 minutes on ice, the ice bath was removed, solid N-hydroxysuccinimide (2 g, 0.017 M) was added (completely dissolved), and immediately afterwards, solid pre-powdered morpholine acetic acid ( Or 1-¹³C or -2-¹³C morpholine acetic acid) (3.64 g, 0.016 M) was added. Morpholine acetic acid gradually dissolved to give a uniform solution that rapidly became cloudy. The reaction was carried out overnight at room temperature with vigorous stirring. The white solid was washed with tetrahydrofuran and vacuum dried (weight 3.65 g (67%), IR spectrum 1828.0 cm-1, 1790.0 cm-1, 1736.0 cm-1).</p><p> (Example 3 Analytical product measurement and relative quantification with two types of samples) Lyophilized Glu-Fibrinopeptide B (Sigma) 100 pmol amount in ice-cooled 0.5 MOPS buffer (pH 7.8 with NaOH) for 30 minutes on ice, I or II (Structure) 1A for preparation, see Examples 1 and 2 for preparation) 200 μl of any newly prepared 2% w / v solution was reacted. TFA was added to a final concentration of 0.5% v / v to terminate the reaction. The modified peptides were then mixed in various predetermined ratios covering approximately the 1:10 to 10: 1 range of the differentially labeled peptides. Millipore C18 Zip-Chip (Millipore C18) Each peptide mixture was individually purified by reverse phase desalting with Zip-Tip). Excess reagents and buffers were not retained on reverse phase packing and were efficiently removed prior to MS analysis. The mixture (0.5 μl) was then spotted onto a MALDI target plate, overspotted with 0.5 μl of 1% w / vα-cyanosilicate skin acid in 50% aqueous acetonitrile, and mounted on a QTOF analyzer. Each sample was analyzed using the source.</p><p> FIG. 2 is an enlarged plot of the MS spectrum obtained from a 1: 1 mixture of Glu-fibrino peptides modified with Reagents I and II. The peak at m / z 1699 represents the N-terminal modified mass of the Glu-fibrino peptide, and as expected, the m / of two different forms of the peptide (see Figures 1A (III) and 1A (IV)). There is no observable difference in z. The modified peptide is isobaric. The isotope clusters observed for the peak are exactly as predicted for a single species.</p><p> A single charged precursor ion of m / z 1699 was then selected for fragmentation with a low energy CID (collision offset of about -70V) to generate the MS / MS spectrum found in Figure 3. As expected, the observed ion sequences were predominantly b-type and y-type. All of these ions were not confirmed to contain a 1: 1 mixture of differentially labeled peptide species and occurred as a single species. For example, the magnified y-ion predominant at m / z 1056.5 is shown in the magnified plot as Figure 4, and the b-ion predominant at m / z 886.3 is shown in the magnified plot as Figure 5.</p><p> However, close examination of the spectrum at about 100 m / z (Fig. 6) reveals the presence of both species VII and VIII (Fig. 1B), which are the fragmentation products of species V and VI (Fig. 1B), respectively. is there. No peaks observed at m / z 128.1 indicate that species V and VI are not stable enough to be observed. Therefore, in this example, fragmentation of the amide bond (eg, bond Y) between the carbonyl group of the peptide and the amino-terminal amino acid results in subsequent fragmentation of the reporter / linker moiety (bond X) and the carbonyl moiety as neutral CO. May cause loss. Peak integration was performed using the instrument's instrumental instrument. After correction for the contribution of about 6% of the naturally occurring second C-13 isotope, the measured relative ratio of VIII / VII (101/100) was 1.03 (predicted value 1.00). Table 1 shows the measured ratio to the observed ratio for the additional experimental mixture prepared by the correction for the naturally occurring second C-13 contribution (ratio expressed as intensity m / z 101 / m / z 100). This data is also represented as a graph in FIG. There is a good match between the observed and predicted values with an average error of &lt;10%.</p><p><tables num="1"><img file="JP2010190902A_D0064.tif" /></tables> (Example 4 Proteome analysis) In practice, a typical proteome analysis can be performed as follows. Whole cell protein extracts for comparison (eg, Samples A and B) are separately digested with trypsin or another proteolytic enzyme. The resulting peptide mixture is separately reacted with different isomer or isobaric labeling reagents (eg, Compounds I and II) to complete the N-terminus of the peptide and lysine amine. For example, sample A can be reacted with compound I and sample B can be reacted with compound II. Each of the samples containing the modified peptide / protein is then mixed together, followed by chromatographic separation (often using multidimensional HPLC) and analysis by MS and MS / MS techniques. When the groups are isobaric, labeling can be performed using a single labeling process (pre-blocking of lysine groups with a second reagent is not required). The labeled protein / peptide mixture is then separated by chromatography and the eluate or fraction thereof analyzed by mass spectrometry as described in Example 3 above. It is also possible to significantly increase the effective sensitivity using a triple quadrupole mass spectrometer or a Q-trap mass spectrometer, where 100 and 101 m / z regions are monitored in precursor ion mode. To do. The relative ratio of the two "signature" peaks is directly correlated with the ratio of each peptide / protein analysis of interest in each of Samples A and B. As used herein, a "signature" peak is a peak for a reporter.</p><p> (Example 5 Analytical product measurement and quantification based on internal standards) Whole cell protein extracts for comparison (eg, Samples A and B) are separately digested with trypsin. The resulting peptide mixture is reacted separately with X and XI (FIG. 8) to substantially completely modify the N-terminus and lysine amine as described above. For example, sample A peptide is reacted with X and sample B peptide is reacted with XI. Then, known amounts or each of Samples A and B containing substantially modified peptides are mixed together. Synthetic peptides that correspond exactly to the combined mixture of A and B with amino acid sequences and / or post-translational modifications (eg, phosphorylation) to the peptides (groups) that may be present in the mixture of samples A and B. A set of (groups) (one or more) is added in exactly the measured amount, where the synthetic peptide (group) is added to another component of the set of homogeneous labeling reagents (eg, compound XII or XIII). , See Figure 8). The combined peptide mixture from Sample A, Sample B, and the synthetic internal reference peptide is optionally chromatographed or electrophoretically separated, for example by multidimensional HPLC, and then by MS and MS / MS techniques. , Can be analyzed as described above. Sample A, Sample B, And all of the equivalent labeled peptides derived from the synthetic counterparts of the same sequence are isobaric and have substantially the same chromatographic properties. By "substantially identical chromatographic property" is meant that little, if any, segregation of differentially labeled but identical peptides is performed. Following MS / MS analysis, the intensity of the reporter due to the reference peptide labeled with an additional component of the isobaric set (eg, XII or XIII) (based on the "signature peak"). By comparing the relative intensities of the reporter to X (Sample A) and XI (Sample B), it is possible to accurately measure the absolute concentration of peptides derived from Samples A and B.</p><p> Although the above is a description of two types of samples (ie, samples A and B), this process can be applied to many practical methods. For example, if there is a sufficiently wide set of labeling reagents, it is possible to analyze a large number of samples at the same time.</p><p> It is possible to have a double (or more than double if there are more samples to analyze) internal criteria (eg, sample A peptide "spiked" with a synthetic peptide labeled with reagent XII. It is possible to "spiked" and the sample B peptide can be spiked with a synthetic peptide labeled with Reagent XIII (at a known absolute concentration). When all are combined, separated and analyzed as described above, the sample A peptide can be quantified relative to the signature peak for compound XII and the sample B peptide can be quantified relative to the signature peak for compound XIII. It can be quantified.</p><p> (Example 6 Typical synthesis of piperidine acetate N-hydroxysuccinimide ester) Bromoacetic acid is dissolved in tetrahydrofuran (or another suitable non-nucleophilic solvent) and added dropwise to a stirred solution containing excess piperidine in tetrahydrofuran (THF, or another suitable non-nucleophilic solvent). To do. The solution is stirred at room temperature for 1-3 days. The solid is filtered, washed with THF (or another suitable non-nucleophilic solvent) and optionally recrystallized. For two different homomeric piperidine acetic acids, 1-¹³C Bromoacetic acid (bromoacetic-1-¹³C acid) (Aldrich PN 27,933-1) or 2-¹³C Bromoacetic acid (bromoacetic-2-¹³Replace any of C acid) (Aldrich PN 27,935-8) with bromoacetic acid. Isomerically substituted piperidines can be prepared from suitable starting materials or are available on a custom order basis from sources such as Cambridge Isotope Laboratories or Isotec.</p><p> Dimethylformamide (DMF) is dissolved in tetrahydrofuran (or another suitable non-nucleophilic solvent) to convert the acetic acid derivative to an active ester such as N-hydroxysuccinimidyl ester. This solution is added dropwise to a stirred solution of equimolar thionyl chloride (based on the molar amount of DMF) dissolved in tetrahydrofuran (or another suitable non-nucleophilic solvent) and cooled in an ice bath. After the addition is complete and 30 minutes on ice, the ice bath is removed, solid N-hydroxysuccinimide is added, and immediately thereafter, piperidine acetic acid (or 1-¹³C or -2-¹³C piperidine acetic acid) is added. Reaction at room temperature overnight with vigorous stirring. The product piperidine acetate N-hydroxysuccinimide ester is then isolated from the reaction mixture, preferably by filtration only. Optionally, recrystallization and / or chromatography can be used to purify the crude product.</p><p> (Example 7 Typical synthesis of piperazine acetate N-hydroxysuccinimide ester) A solution containing 2 equivalents of piperazine dissolved in tetrahydrofuran (THF) is added dropwise to a solution containing 1 equivalent of bromoacetic acid (relative to the amount of piperazine) dissolved in tetrahydrofuran. The two solutions should be concentrated to be practical. The resulting reaction solution is stirred at room temperature for 1-3 days. The solid is filtered, washed with THF and optionally recrystallized. For two different homomeric piperidine acetic acids, 1-¹³C Bromoacetic acid (bromoacetic-1-¹³C acid) (Aldrich PN 27,933-1) or 2-¹³C Bromoacetic acid (bromoacetic-2-¹³Any of C acid) (Aldrich PN 27,935-8) can be used in place of bromoacetic acid.</p><p> Dry dimethylformamide (DMF, 1.75 g, 0.024 M) can be dissolved in tetrahydrofuran to convert the acetic acid derivative to an active ester such as N-hydroxysuccinimidyl ester. This solution can be added dropwise to a stirred solution of equimolar thionyl chloride (based on the molar amount of DMF) dissolved in tetrahydrofuran and cooled in an ice bath. After the addition is complete and 30 minutes on ice, the ice bath is removed, solid N-hydroxysuccinimide is added, and immediately thereafter, piperazine acetic acid (or 1-¹³C or -2-¹³C piperidine acetic acid) can be added. The reaction can be carried out overnight at room temperature with vigorous stirring. The product piperazine acetate N-hydroxysuccinimide ester can then be isolated from the reaction mixture, if possible only by filtration. The crude product can then be purified using recrystallization and / or chromatography.</p><p> (Example 8 Typical synthesis of N, N'-(2-methoxyethyl) -glycine active ester (transcription from US Pat. No. 6,326,479)) 500 mmol of tert-butylchloroacetate (Aldrich Chemical) was added dropwise to 1.1 mole of bis (2-methoxyethyl) amine (Aldrich Chemical). The reaction was stirred for 3 days and then the reaction was post-treated. 250 mL of dichloromethane (DCM) and 200 mL of water were added to the final reaction contents. In this stirred solution, solid potassium carbonate (K)₂CO₃) 300 mmol was added little by little. After mixing was complete, the layers were separated. Wash the DCM layer once with 1 volume of water and allow it to dry (Na₂SO₄), Filtered and evaporated to give 66.3 g of very pale yellow oil. The crude product was distilled in Kugelrohr at 60 ° C. (200-500 μM Hg) to give 58.9 g (238 mmol, 95%) of a clear, colorless oil.</p><p> 12.1 mL of concentrated hydrochloric acid was gradually added to the purified (stirred) N, N'-(2-methoxyethyl) -glycine-tert-butyl ester. The mixture was stirred overnight and reacted, after which by-products (eg water, HCl, isobutylene) were removed by vacuum evaporation.¹H-MNR analysis showed that the t-butyl ester was hydrolyzed, but water and HCl were still considered to be present. The crude product was co-evaporated twice from acetonitrile (ACN), but water and HCl were still present. To remove contaminants, 4.4 g aliquots were removed from the crude product and distilled in Kugelrohr at 135-155 ° C (100-200 μM Hg, where the pressure drops rapidly after the start of distillation). Yield 4.2 g (18.4 mmol, 95% recovery of thick colorless clear oil). The distilled product contained no water or HCl.</p><p> An active ester of any suitable isotope-labeled substituted or unsubstituted N, N'-(2-methoxyethyl) -glycine (eg, N-hydroxysuccinimidyl ester) is then incorporated herein by reference. It can be prepared by a method known in the art such as the method described.</p><p> (Example 9 Typical method for preparing a solid support containing a labeling / tagging reagent) Commercially available peptide synthesis resins containing "sterically hindered cleavable linkers" are mixed with at least a 2-fold excess of aminoalkylpiperazine (eg, 1- (2-aminoethyl) piperazine, Aldrich P / N A5,520-9, isomer forms can be made by the process described in FIG. 11 in combination with the description herein). A "sterically hindered cleavable linker" is one in which the linker forms a cleavable covalent bond between a solid support and an atom or group that reacts with the cleavable linker. It means that it contains secondary or tertiary atoms. Non-limiting examples of sterically hindered solid supports include trityl chloride resin (Trityl-Cl, Novabiochem, P / N 01-64-0074), 2-chlorotrityl chloride resin (Novabiochem, P / N). 01-64-0021), DHPP (Bachem, P / N Q-1755), MBHA (Applied Biosystems P / N 400377), 4-Methyltrityl chloride resin (Novabiochem, P / N 01-64-0075), 4- Methoxytrityl chloride resin (Novabiochem, P / N 01-64-0076), hydroxy- (2-chlorophenyl) methyl-PS (Novabiochem, P / N 01-64-0345), Rink Acid Resin (Rink Acid Resin) Novabiochem, P / N 01-64-0380 and 01-64-0202), Novabiochem, P / N TGT (NovaSyn TGT) Alcohol Resin (Novabiochem, P / N) 01-64-0074). The excess reagent is then removed by washing the support. The secondary amine of the support-bound piperazine is then reacted with excess bromoacetic acid in the presence of a tertiary amine such as triethylamine. The excess reagent is then removed by washing the support. Support-linked carboxylic acid groups of bis-alkylated piperazine, depending on the method used to produce the active ester of the carboxylic acid (eg, whether or not a salt of the carboxylic acid is required for the synthesis of the active ester). The wash can be selected to have a pH adjusted to protonate the. The carboxylic acid group of the support-bound piperazine is then subjected to an active ester (eg, N-hydroxysk) using procedures known in the art for the production of the active ester of the carboxylic acid, such as the methods described above. Convert to synimidyl ester). Then, the obtained solid support can be used to label an analysis product (for example, a peptide) of a sample having a nucleophilic functional group. The labeled analyte can then be released from the support as described in the manufacturer's product instructions. The products of each cleavage reaction can then be combined to form a sample mixture.</p>