Insulin receptor substrate 1 (irs1) protein srm/mrm assay

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DESCRIPTION

Insulin Receptor Substrate 1 Protein (IRS1) SRM / MRM Assay

Field of the invention

The present invention relates to a method for measuring the level of insulin receptor substrate protein 1 (IRS1) in a human biological sample of formalin-fixed tissue.

Introduction

Specific peptides derived from subsequences of insulin receptor substrate protein 1 are described, and will be referred to as IRS1. The peptide sequence and fragmentation / transition ions for each peptide are especially useful in a mass spectrometry-based selected reaction monitoring (SRM), which may also be referred to as a multiple reaction monitoring (MRM) assay, and will be referred to as SRM. / MRM. Information on the use of peptides for quantitative SRM / MRM analysis of IRS1 protein is described.

This SRM / MRM assay can be used to measure the relative or absolute quantitative levels of one or more of the specific peptides of the IRS1 protein, and thus provides a means of measuring the amount of the IRS1 protein in a particular preparation of proteins obtained from a biological sample by mass spectrometry.

More specifically, the SRM / MRM assay can measure these peptides directly in samples of complex protein lysates prepared from cells obtained from patient tissue samples, such as formalin-fixed cancer patient tissues. Methods for preparing protein samples from formalin-fixed tissue are described in US Patent No. 7,473,532. The methods described in US Pat. No. 7,473,532 can be conveniently carried out using Liquid Tissue ™ reagents and the protocol available from Expression Pathology Inc. (Rockville, MD).

The most widely and advantageously available form of cancer patient tissue is formalin-fixed, paraffin-embedded tissue. Formaldehyde / formalin fixation of surgically removed tissue is by far the most common method of preserving cancerous tissue specimens worldwide, and is the accepted convention for routine practice in pathology. Aqueous solutions of formaldehyde are called formalin. 100% formalin consists of a saturated solution of formaldehyde (i.e., about 40% by volume or 37% by mass) in water, with a small amount of stabilizer, usually methanol, to limit oxidation and the degree of polymerization. The most common way to preserve tissue is to soak all tissue for extended periods of time (8 hours to 48 hours) in aqueous formaldehyde, commonly referred to as 10% neutral buffered formalin, followed by embedding all of the fixed tissue in paraffin wax for long-term storage at room temperature. Thus, molecular analytical methods for analyzing formalin-fixed cancerous tissue will be the most widely accepted and widely used methods for analyzing cancer patient tissues.

Results from the SRM / MRM assay can be used to correlate accurate and precise quantitative levels of IRS1 protein in specific tissue samples (eg, cancer tissue sample) from the patient or subject from which it was collected and preserved. the tissue (biological sample). This not only provides diagnostic information about the cancer, but also allows a physician or other medical professional to determine the appropriate therapy for the patient. Such an assay, which provides important diagnostic and therapeutic information on protein expression levels in diseased tissue or other sample from the patient, is called an adjunct diagnostic assay. For example, such an assay can be designed to diagnose the stage or grade of a cancer and to determine a therapeutic agent to which a patient is most likely to respond.

Yi Z et al. (J Am Soc Mass Spectrom 2006, 17: 562-567) reports on the quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS / Ms.

Summary

The underlying problem of the present invention is solved by the subject matter of the appended independent claims. The preferred embodiments of the dependent claims can be taken.

More specifically, the underlying problem of the present invention is solved by a method of measuring the level of insulin receptor substrate protein 1 (IRS1) in a human biological sample of formalin-fixed tissue, which comprises detecting and / or quantifying the amount of an IRS1 peptide fragment in a protein digest prepared from said human biological sample by using mass spectrometry; and calculating the level of IRS1 protein in said sample;

wherein the peptide fragment of IRS1 is SEQ ID NO: 70 or SEQ ID NO: 76, and wherein said level is a relative level or an absolute level.

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In one embodiment, the method further comprises the step of fractionating said protein digest prior to detecting and / or quantifying the amount of said IRS1 peptide fragment.

In one embodiment, said protein digest comprises a protease digest.

In one embodiment, the tissue is paraffin embedded tissue.

In one embodiment, the tissue is obtained from a tumor.

In one embodiment, the method further comprises quantifying said IRS1 peptide fragment.

In one embodiment, quantifying said IRS1 peptide fragment comprises comparing the amount of said IRS1 peptide fragment in a biological sample with the amount of the same IRS1 peptide fragment in a separate and different biological sample.

In one embodiment, the quantification of said IRS1 peptide fragment comprises determining the amount of said IRS1 peptide fragment in a biological sample by comparison with an internal standard peptide added in a known amount, wherein said IRS1 peptide fragment in the biological sample is compared to an internal standard peptide having the same amino acid sequence; and wherein the internal standard peptide is an isotopically labeled peptide.

In one embodiment, detection and / or quantification of the amount of said IRS1 peptide fragment in the protein digest indicates the presence of the modified or unmodified IRS1 protein and an association with cancer in the subject.

In one embodiment, the method further comprises correlating the results of said detection and / or quantification of the amount of said IRS1 peptide fragment, or the level of said IRS1 protein with the diagnosis of the stage / grade / status of the cancer.

In one embodiment, the correlation of the results of said detection and / or quantification of the amount of said IRS1 peptide fragment, or the level of said IRS1 protein with the diagnosis of the stage / grade / status of the cancer is combined with the detection and / or quantification of the amount of other proteins or peptides of other proteins in a multiplexed format to provide additional information on the diagnosis of the stage / grade / status of the cancer.

The assays described herein measure the relative or absolute levels of IRS1 protein-specific unmodified peptides and can also measure the absolute or relative levels of IRS1 protein-specific modified peptides. Examples of modifications include phosphorylated amino acid residues and glycosylated amino acid residues that are present in peptides.

The relative quantitative levels of the IRS1 protein are determined using the SRM / MRM methodology, for example by comparing the areas of the distinctive SRM / MRM peaks (e.g., the area of ​​the distinctive peaks or the integrated intensity of the ions fragments) of a single IRS1 peptide in different samples. Alternatively, it is possible to compare multiple SRM / MRM signature peak areas for multiple IRS1 signature peptides, where each peptide has its own specific SRM / MRM signature peak, to determine the relative content of IRS1 protein in a sample. biological with the IRS1 protein content in one or more additional or different biological samples. In this way, the amount of a particular peptide, or peptides, of the IRS1 protein, and therefore the amount of the IRS1 protein, relative to the same IRS1 peptide, or peptides, in 2 or more biological samples in the same experimental conditions. In addition, the relative quantification for a particular peptide, or peptides, of the IRS1 protein in a single sample can be determined by comparing the area of ​​the distinctive peak for that peptide using the SRM / MRM methodology versus the area of ​​the distinctive peak for a different peptide. , or peptides, of a different protein, or proteins, in the same protein preparation of the biological sample. In this way, the amount of a particular peptide of the IRS1 protein, and therefore the amount of the IRS1 protein, is determined with respect to another in the same sample. These approaches generate the quantification of an individual peptide, or peptides, of the IRS1 protein with respect to the amount of another peptide, or peptides, between samples and within samples, in which the amounts determined by the area of ​​the peaks are relative between yes, regardless of the absolute amounts of weight for volume or weight for weight of the IRS1 peptide in the protein preparation of the biological sample. The relative quantitative data on the individual distinctive peak areas between different samples is normalized to the amount of protein analyzed per sample. Relative quantification can be carried out simultaneously with many multi-protein peptides and the IRS1 protein in a single sample and / or with many samples to better understand the relative amounts of proteins, one peptide / protein relative to other peptides / proteins.

The absolute quantitative levels of the IRS1 protein are determined, for example, by the SRM / MRM methodology, whereby the area of ​​the distinctive SRM / MRM peak of an individual peptide of the IRS1 protein in a biological sample is compared to the SRM / MRM distinctive peak area of ​​an added internal standard. In one embodiment, the internal standard is a synthetic version of exactly the same IRS1 peptide that contains one or

ES 2 642 807 T3 plus amino acid residues labeled with one or more heavy isotopes. Such isotope-labeled internal standards are synthesized such that, when analyzed by mass spectrometry, a consistent and predictable SRM / MRM signature peak is generated that is different and distinct from the native IRS1 peptide signature peak and can be used. as a comparative peak. Thus, when the internal standard is added to a protein preparation from a biological sample in known amounts and analyzed by mass spectrometry, the area of ​​the signature SRM / MRM peak of the native peptide is compared to the area of ​​the signature SRM peak. / MRM of the internal standard peptide, and this numerical comparison indicates the absolute molarity and / or the absolute weight of the native peptide present in the original protein preparation of the biological sample. The absolute quantitative data for the peptide fragments are expressed according to the amount of protein analyzed per sample. Absolute quantification with many peptides, and therefore proteins, can be carried out simultaneously on a single sample and / or with many samples to better understand the absolute amounts of proteins in individual biological samples and in complete cohorts of individual samples.

The SRM / MRM assay method can be used to aid in the diagnosis of the stage of cancer, for example, directly on tissue obtained from a patient, such as formalin-fixed tissue, and to help determine which therapeutic agent would be the most advantageous for use in treating that patient. Cancerous tissue that is removed from a patient by means of surgery, such as for the therapeutic removal of partial or complete tumors, or by means of biopsy procedures carried out to determine the presence or absence of a presumed disease, is analyzed for determine whether or not a specific protein, or proteins, is present, and what forms of proteins are present in that patient tissue. In addition, the expression level of a protein, or multiple proteins, can be determined and can be compared to a normal or reference level found in healthy tissue. The normal or reference levels of proteins found in healthy tissue can be obtained, for example, from the relevant tissues of one or more individuals who do not have cancer. Alternatively, normal or reference levels for individuals with cancer can be obtained by analyzing relevant tissues unaffected by cancer. Assays of protein levels (eg, IRS1 levels) can also be used to diagnose the stage of cancer in a patient or subject diagnosed with cancer using IRS1 levels. The levels or amounts of proteins or peptides can be defined as the amount expressed in moles, mass or weight of a protein or peptide determined by the SRM / MRM assay. The level or amount can be normalized to the total level or amount of protein or other component in the lysate tested (eg, expressed in micromoles / microgram of protein or micrograms / micrograms of protein). Furthermore, the level or amount of a protein or peptide can be determined relative to volume, expressed, for example, in micromolar or nanograms / microliter. The level or amount of protein or peptide determined by the SRM / MRM assay can also be normalized to the number of cells analyzed. Thus, information regarding IRS1 can be used to help determine the stage or grade of a cancer by correlating the level of IRS protein (or peptide fragments of IRS1 protein) with levels observed in normal tissues. Once the stage and / or grade, and / or expression characteristics of the cancer IRS1 protein have been determined, that information can be compared to a list of therapeutic agents (chemical and biological) developed to specifically treat cancer. cancerous tissue characterized, for example, by abnormal expression of the protein or protein (s) (eg, IRS1) that were tested. Comparison information from an IRS1 protein assay to a list of therapeutic agents that specifically select, for example, the IRS1 protein or cells / tissues expressing the protein, defines what has been termed a personalized medicine approach to treat disease. The assay methods described herein form the basis of a personalized medicine approach using the analysis of proteins from the patient's own tissue as a source for diagnostic and treatment decisions.

Detailed description

In principle, any predicted peptide derived from the IRS1 protein, prepared for example by digestion with a protease of known specificity (eg trypsin), can be used as a surrogate indicator to determine the abundance of the IRS1 protein in a sample by the use of an SRM / MRM assay based on mass spectrometry. Similarly, any predicted peptide sequence containing an amino acid residue at a site known to be potentially modified in the IRS1 protein could also potentially be used to test the degree of modification of the IRS1 protein in a sample.

Peptide fragments of IRS1 can be generated by a variety of means, including the use of the Liquid Tissue ™ protocol provided in US Patent 7,473,532. The Liquid Tissue ™ protocol and reagents are capable of producing peptide samples suitable for mass spectroscopic analysis from formalin-fixed paraffin-embedded tissue by proteolytic digestion of tissue / biological sample proteins. In the Liquid Tissue ™ protocol, the tissue / biological sample is heated in a buffer for an extended period of time (e.g., from about 80 ° C to about 100 ° C for a period of time of about 10 minutes to about 4 hours) to reverse or release protein cross-linking. The buffer used is a neutral buffer, (eg, a Tris-based buffer, or a buffer containing a detergent). Following heat treatment, the tissue / biological sample is treated with one or more proteases, including, but not limited to, trypsin, chymotrypsin, pepsin, and endoproteinase Lys-C for a time sufficient to alter the tissue and cellular structure of said tissue. biological sample, and to liquefy said sample (eg, a period of time from 30 minutes to 24 hours at a temperature of 37 ° C to 65 ° C). The result of heating and proteolysis is a dilute, soluble, liquid biomolecule lysate.

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Surprisingly, many potential IRS1 protein peptide sequences were found to be unsuitable or ineffective for use in mass spectrometry-based SRM / MRM assays for reasons that are not immediately apparent. As it was not possible to predict the most suitable peptides for the MRM / SRM assay, it was necessary to experimentally identify modified and unmodified peptides in actual Liquid Tissue ™ lysates to develop a reliable and accurate SRM / MRM assay for the IRS1 protein. Although not wishing to be bound by any theory, it is believed that certain peptides, for example, could be difficult to detect by mass spectrometry as they do not ionize well or produce different fragments from other proteins, peptides may also not resolve well in separation (eg, liquid chromatography), or can adhere to glass or plastic material.

The IRS1 peptides of this disclosure (eg, Tables 1 and 2) were obtained from the IRS1 protein by protease digestion of all proteins in a Liquid Tissue ™ complex lysate prepared from cells obtained from fixed cancer tissue. in formalin. Unless otherwise indicated, in each case the protease was trypsin. The Liquid Tissue ™ lysate was then analyzed by mass spectrometry to determine those peptides derived from the IRS1 protein that are detected and analyzed by mass spectrometry. The identification of a specific preferred subgroup of peptides for mass spectrometric analysis is based on; 1) the experimental determination of which peptide (s) of a protein ionize in mass spectrometry analyzes of Liquid Tissue ™ lysates, and 2) the ability of the peptide to survive the protocol and the experimental conditions used to prepare a lysate of Liquid Tissue ™. This latter property extends not only to the amino acid sequence of the peptide, but also to the ability of a modified amino acid residue within a peptide to survive in modified form during sample preparation.

Table 1

<td>Table 1 SEQ ID No.</td><td>Peptide Sequence</td>

<td>SEQ ID N °: 1</td><td>EVWQVILKPKGLGQTK</td>

<td>SEQ ID N °: 2</td><td>GLGQTKNLIGIYRLCLTSK</td>

<td>SEQ ID No: 3</td><td>GSGDYMPMSPKSVSAPQQIINPIR</td>

<td>SEQ ID No: 4</td><td>LCGAAGGLENGLNYIDLDLVK</td>

<td>SEQ ID No: 5</td><td>LNSEAAAWLQLMNIRR</td>

<td>SEQ ID No: 6</td><td>LWTNGVGGHHSHVLPHPK</td>

<td>SEQ ID No: 7</td><td>NKHLVALYTR</td>

<td>SEQ ID No: 8</td><td>PKGLGQTKNLIGIYR</td>

<td>SEQ ID No: 9</td><td>RSIPLESCFNINK</td>

<td>SEQ ID No: 10</td><td>RTHSAGTSPTITHQK</td>

<td>SEQ ID No: 11</td><td>SQSSSNCSNPISVPLRRHHLNNPPPSQVGLTR</td>

<td>SEQ ID No: 12</td><td>SVSAPQQIINPIRR</td>

<td>SEQ ID No: 13</td><td>TISFVKLNSEAAAWLQLMNIR</td>

<td>SEQ ID No: 14</td><td>VDTAAQTNSRLAR</td>

<td>SEQ ID No: 15</td><td>VIRADPQGCRR</td>

<td>SEQ ID No: 16</td><td>AASEAGGPARLEYYENEK</td>

<td>SEQ ID No: 17</td><td>AAWQESTGVEMGR</td>

<td>SEQ ID No: 18</td><td>AAWQESTGVEMGRLGPAPPGAASICR</td>

<td>SEQ ID No: 19</td><td>ADPQGCR</td>

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<td>Table 1 SEQ ID No.</td><td>Peptide Sequence</td>

<td>SEQ ID No: 20</td><td>AMSDEFRPRSK</td>

<td>SEQ ID No: 21</td><td>AREQQQQQQPLLHPPEPK</td>

<td>SEQ ID No: 22</td><td>ASSDGEGTMSRPASVDGSPVSPSTNR</td>

<td>SEQ ID No: 23</td><td>CPSQLQPAPR</td>

<td>SEQ ID No: 24</td><td>EEETGTEEYMK</td>

<td>SEQ ID No: 25</td><td>CTPGTGLGTSPALAGDEAASAADLDNR</td>

<td>SEQ ID No: 26</td><td>MDLGPGRR</td>

<td>SEQ ID No: 27</td><td>FFVLRAASEAGGPAR</td>

<td>SEQ ID No: 28</td><td>GGNGHRCTPGTGLGTSPALAGDEAASAADLDNR</td>

<td>SEQ ID No: 29</td><td>HHLNNPPPSQVGLTR</td>

<td>SEQ ID No: 30</td><td>HSAFVPTRSYPEEGLEMHPLER</td>

<td>SEQ ID No: 31</td><td>GSGDYMPMSPK</td>

<td>SEQ ID No: 32</td><td>VDTAAQTNSR</td>

<td>SEQ ID No: 33</td><td>KVGYLRK</td>

<td>SEQ ID No: 34</td><td>LARPTRLSLGDPK</td>

<td>SEQ ID No: 35</td><td>LHPPLNHSRSIPMPASRCSPSATSPVSLSSSSTSGHGSTSDCLFPR</td>

<td>SEQ ID No: 36</td><td>LLYAATADDSSSSTSSDSLGGGYCGAR</td>

<td>SEQ ID No: 37</td><td>LSLGDPKASTLPR</td>

<td>SEQ ID No: 38</td><td>LSTSSGR</td>

<td>SEQ ID No: 39</td><td>PASVDGSPVSPSTNRTHAHR</td>

<td>SEQ ID No: 40</td><td>PDSSTLHTDDGYMPMSPGVAPVPSGR</td>

<td>SEQ ID No: 41</td><td>PGELGGAPK</td>

<td>SEQ ID No: 42</td><td>PRSKSQSSSNCSNPISVPLR</td>

<td>SEQ ID No: 43</td><td>PTRLSLGDPKASTLPR</td>

<td>SEQ ID No: 44</td><td>QSYVDTSPAAPVSYADMR</td>

<td>SEQ ID N °: 45</td><td>RHHLNNPPPSQVGLTR</td>

<td>SEQ ID No: 46</td><td>HSSETFSSTPSATR</td>

<td>SEQ ID No: 47</td><td>RSRTESITATSPASMVGGK</td>

<td>SEQ ID No: 48</td><td>RSSEDLSAYASISFQK</td>

<td>SEQ ID No: 49</td><td>SIPLESCFNINK</td>

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<td>Table 1 SEQ ID No.</td><td>Peptide Sequence</td>

<td>SEQ ID No: 50</td><td>SKSQSSSNCSNPISVPLR</td>

<td>SEQ ID No: 51</td><td>SRTESITATSPASMVGGK</td>

<td>SEQ ID No: 52</td><td>SSASVSGSPSDGGFISSDEYGSSPCDFR</td>

<td>SEQ ID No: 53</td><td>SSEDLSAYASISFQKQPEDR</td>

<td>SEQ ID No: 54</td><td>SSFRSVTPDSLGHTPPA</td>

<td>SEQ ID No: 55</td><td>GEEELSNYICMGGK</td>

<td>SEQ ID No: 56</td><td>SVTPDSLGHTPPAR</td>

<td>SEQ ID No: 57</td><td>SYPEEGLEMHPLER</td>

<td>SEQ ID No: 58</td><td>TESITATSPASMVGGK</td>

<td>SEQ ID No: 59</td><td>VGNTVPFGAGAAVGGGGGSSSSSEDVK</td>

<td>SEQ ID N °: 60</td><td>VNLSPNRNQSAK</td>

<td>SEQ ID No: 61</td><td>GSGDYMPMSPK</td>

<td>SEQ ID No: 62</td><td>ASSDGEGTMSRPASVDGSPVSPSTNR</td>

<td>SEQ ID No: 63</td><td>SVSAPQQIINPIR</td>

<td>SEQ ID No .: 64</td><td>LCLTSKTISFVKLNSEAAAVVLQLMNIR</td>

<td>SEQ ID No: 65</td><td>LEPSLPHPHHQVLQPHLPR</td>

<td>SEQ ID No .: 66</td><td>LPGHRHSAFVPTR</td>

<td>SEQ ID No: 67</td><td>SSEDLSAYASISFQK</td>

<td>SEQ ID No: 68</td><td>PDSSTLHTDDGY [phosphoryl] MPMSPGVAPVPSGR</td>

<td>SEQ ID No: 69</td><td>SPGEY [phosphoryl] VNIEFGSDQSGYLSGPVAFHSSPSVR</td>

<td>SEQ ID No: 70</td><td>EQQQQQQPLLHPPEPK</td>

<td>SEQ ID No: 71</td><td>HSSASFENVWLRPGELGGAPK</td>

<td>SEQ ID No: 72</td><td>LEYYENEK</td>

<td>SEQ ID No: 73</td><td>LNSEAAAVVLQLMNIR</td>

<td>SEQ ID No: 74</td><td>LSLGDPK</td>

<td>SEQ ID No: 75</td><td>NLIGIYR</td>

<td>SEQ ID No .: 76</td><td>TGIAAEEVSLPR</td>

<td>SEQ ID No: 77</td><td>HLVALYTR</td>

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Table 2

<td>Table 2 SEQ ID No.</td><td>Peptide sequence</td><td>Mass Monoisotopic</td><td>Precursor State of Charge</td><td>precursor m / z</td><td>transition m / z</td><td>Ion Type</td>

<td>SEQ ID N °: 22</td><td>ASSDGEGTMSRPASVDGSPV 5P5TNR</td><td> 2548,146</td><td> 2</td><td> 1275,07996</td><td> 574,2938</td><td>y5</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 857,447</td><td>y8</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1302,628</td><td>y13</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1373,665</td><td>y14</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1470,718</td><td>y15</td>

<td></td><td></td><td></td><td> 3</td><td> 850,388977</td><td> 944,4791</td><td>y9</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1001,5</td><td>y10</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1116,527</td><td>y11</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1215,596</td><td>y12</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1302,628</td><td>y13</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1373,665</td><td>y14</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1470,718</td><td>y15</td>

<td>SEQ ID N °: 70</td><td>EQQQQQQPLLHPPEPK</td><td> 1923,98</td><td> 2</td><td> 962,997009</td><td> 930,5402</td><td>y8</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1027,593</td><td>y9</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1155,651</td><td>y10</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1283,71</td><td>y11</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1411,769</td><td>y12</td>

<td></td><td></td><td></td><td> 3</td><td> 642,333984</td><td> 578,3294</td><td>y10</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 704,3721</td><td>y6</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 706,388</td><td>y12</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 770,4172</td><td>y13</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 817,4561</td><td>y7</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 930,5402</td><td>y8</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1027,593</td><td>y9</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1155,651</td><td>y10</td>

<td>SEQ ID N °: 77</td><td>HLVALYTR</td><td> 971,555</td><td> 2</td><td> 486,783997</td><td> 552,3135</td><td>y4</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 623,3506</td><td>y5</td>

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<td>Table 2 SEQ ID No.</td><td>Peptide sequence</td><td>Mass Monoisotopic</td><td>Precursor State of Charge</td><td>precursor m / z</td><td>transition m / z</td><td>Ion Type</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 722,419</td><td>y6</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 835,5031</td><td>y7</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 972,562</td><td>y8</td>

<td>SEQ ID N °: 71</td><td>HSSASFENVWLRPGELGGAPK</td><td> 2238,118</td><td> 2</td><td> 1120,06604</td><td> 825,4459</td><td>y9</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1280,71</td><td>y12</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1379,779</td><td>y13</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1493,822</td><td>y14</td>

<td></td><td></td><td></td><td> 3</td><td> 747,046021</td><td> 825,4459</td><td>y9</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 981,5471</td><td>y10</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1008,021</td><td>y19</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1094,631</td><td>y11</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1280,71</td><td>y12</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1379,779</td><td>y13</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1493,822</td><td>y14</td>

<td>SEQ ID N °: 72</td><td>LEYYENEK</td><td> 1086,487</td><td> 2</td><td> 544,25</td><td> 390,1978</td><td>y3</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 682,3037</td><td>y5</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 845,367</td><td>y6</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 974,4096</td><td>y7</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1087,494</td><td>y8</td>

<td>SEQ ID N °: 73</td><td>LNSEAAAVVLQLMNIR</td><td> 1740,956</td><td> 2</td><td> 871,484985</td><td> 774,4285</td><td>y6</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 855,4565</td><td>b8</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 887,5126</td><td>y7</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 986,581</td><td>y8</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1085,649</td><td>y9</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1156,687</td><td>y10</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1227,724</td><td>y11</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1298,761</td><td>y12</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1427,803</td><td>y13</td>

ES 2 642 807 T3

<td>Table 2 SEQ ID No.</td><td>Peptide sequence</td><td>Mass Monoisotopic</td><td>Precursor State of Charge</td><td>precursor m / z</td><td>transition m / z</td><td>Ion Type</td>

<td>SEQ ID N °: 74</td><td>LSLGDPK</td><td> 728,407</td><td> 2</td><td> 365,209992</td><td> 416,2134</td><td>y4</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 529,2975</td><td>y5</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 616,3295</td><td>y6</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 729,4136</td><td>y7</td>

<td></td><td></td><td></td><td> 3</td><td> 354,187012</td><td> 406,2039</td><td>y4</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 507,2516</td><td>y5</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 594,2836</td><td>y6</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 707,3677</td><td>y7</td>

<td>SEQ ID N °: 75</td><td>NLIGIYR</td><td> 847,492</td><td> 2</td><td> 424,752991</td><td> 451,2658</td><td>y3</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 508,2873</td><td>y4</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 621,3713</td><td>y5</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 734,4554</td><td>y6</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 848,4983</td><td>y7</td>

<td>SEQ ID N °: 44</td><td>QSYVDTSPAAPVSYADMR</td><td> 1956,889</td><td> 2</td><td> 979,450989</td><td> 938,4395</td><td>y8</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1009,477</td><td>y9</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1080,514</td><td>y10</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1177,567</td><td>y11</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1264,599</td><td>y12</td>

<td></td><td></td><td></td><td> 3</td><td> 653,302979</td><td> 655,2863</td><td>y5</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 742,3183</td><td>y6</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 841,3867</td><td>y7</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 938,4395</td><td>y8</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1009,477</td><td>y9</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1080,514</td><td>y10</td>

<td></td><td></td><td></td><td> 3</td><td></td><td> 1177,567</td><td>y11</td>

<td>SEQ ID N °: 63</td><td>SVSAPQQIINPIR</td><td> 1421,799</td><td> 2</td><td> 711,906006</td><td> 385,2552</td><td>y3</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 499,2982</td><td>y4</td>

ES 2 642 807 T3

<td>Table 2 SEQ ID No.</td><td>Peptide sequence</td><td>Mass Monoisotopic</td><td>Precursor State of Charge</td><td>precursor m / z</td><td>transition m / z</td><td>Ion Type</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 725,4663</td><td>y6</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 853,5248</td><td>y7</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 981,5834</td><td>y8</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1078,636</td><td>y9</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1149,673</td><td>y10</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1236,705</td><td>y11</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1335,774</td><td>y12</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1422,806</td><td>y13</td>

<td>SEQ ID N °: 57</td><td>SYPEEGLEMHPLER</td><td> 1685,772</td><td> 2</td><td> 843,893005</td><td> 514,2979</td><td>y4</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 718,8453</td><td>y12</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 911,4398</td><td>y7</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1024,524</td><td>y8</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1081,545</td><td>y9</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1210,588</td><td>y10</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1339,63</td><td>y11</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1436,683</td><td>y12</td>

<td>SEQ ID N °: 76</td><td>TGIAAEEVSLPR</td><td> 1241,662</td><td> 2</td><td> 621,838013</td><td> 272,1712</td><td>y2</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 700,3983</td><td>y6</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 829,4409</td><td>y7</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 900,478</td><td>y8</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 971,5151</td><td>y9</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1084,599</td><td>y10</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1141,621</td><td>y11</td>

<td></td><td></td><td></td><td> 2</td><td></td><td> 1242,668</td><td>y12</td>

Cell protein lysates obtained directly from formalin-fixed tissue (formaldehyde) were prepared using the reagents and the Liquid Tissue ™ protocol, which involves collecting the cells into a sample tube by means of tissue microdissection, followed by heating the cells in the Liquid 5 Tissue ™ buffer for an extended period of time. Once formalin-induced cross-linking has been adversely affected, tissue / cells are digested to completion in a predictable manner using a protease, including for example, but not limited to, trypsin protease. Each protein lysate is converted into a collection of peptides by digestion of the intact polypeptides with the protease. Each Liquid Tissue ™ lysate was analyzed (eg, by ion trap mass spectrometry) to perform

ES 2 642 807 T3 multiple global proteomic studies of the peptides, in which the data were presented as the identification of as many peptides as could be identified by mass spectrometry of all the cellular proteins present in each protein lysate. An ion trap mass spectrometer or other form of mass spectrometer that is capable of carrying out a global profile is employed for the identification of as many peptides as possible from a single complex protein / peptide lysate. Ion trap mass spectrometers, however, may be the best type of mass spectrometer for carrying out global peptide profiling. Although the SRM / MRM assay can be developed and performed on any type of mass spectrometer, including a MALDI, ion trap, or triple quadrupole, it is often considered the most advantageous instrument platform for an assay. SRM / MRM is a triple quadrupole instrument platform.

Once as many peptides as possible were identified in a single MS analysis of a single lysate under the conditions employed, that list of peptides was sorted and used to determine the proteins that were detected in that lysate. That process was repeated for multiple Liquid Tissue ™ lysates, and the very long list of peptides was sorted down to a single data set. This type of data set can be considered to represent the peptides that can be detected in the type of biological sample that was analyzed (after digestion with protease), and specifically in a Liquid Tissue ™ lysate of the biological sample, and thus includes peptides for specific proteins, such as, for example, the IRS1 protein.

IRS1 tryptic peptides identified as useful in determining absolute or relative amounts of the IRS1 receptor include one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of the peptides of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID N °: 32, SEQ ID N °: 33, SEQ ID N °: 34, SEQ ID N °: 35, SEQ ID N °: 36, SEQ ID N °: 37, SEQ ID N °: 38, SEQ ID N : 38, SEQ ID No: 40, SEQ ID No: 41, SEQ ID No: 42, SEQ ID No: 43, SEQ ID No: 44, SEQ ID No: 45, SEQ ID No: : 46, SEQ ID No: 47, SEQ ID No: 48, SEQ ID No: 49, SEQ ID No: 50, SEQ ID No: 51, SEQ ID No: 52, SEQ ID No: 53, SEQ ID No: 54, SEQ ID No: 55, SEQ ID No: 56, SEQ ID No: 57, SEQ ID No: 58, SEQ ID No: 59, SEQ ID No: 60, SEQ ID No: 61, SEQ ID No: 62, SEQ ID No: 63, SEQ ID No: 64, SEQ ID No: 65, SEQ ID No: 66, SEQ ID NO: 67, SEQ ID No: 68, SEQ ID No: 69, SEQ ID No: 70, SEQ ID No: 71, SEQ ID No: 72, SEQ ID No: 73, SEQ ID N : 74, SEQ ID No.: 75, SEQ ID No.: 76, and SEQ ID No.: 77, each of which are listed in Table 1. Each of these peptides was detected by mass spectrometry in Liquid Tissue ™ lysates prepared from formalin-fixed, paraffin-embedded tissue. Thus, each of the peptides in Table 1, or any combination of those peptides (eg, one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of those peptides listed in Table 1, and in particular combinations with one or more of the peptides found in Table 2) are candidates for use in the quantitative SRM / MRM assay of the IRS1 protein in human biological samples, that directly include formalin-fixed tissue from a patient.

The IRS1 tryptic peptides listed in Table 1 include those detected from multiple Liquid Tissue ™ lysates from multiple different formalin-fixed tissues from different human organs including prostate, colon, and breast. Each of these peptides is considered useful for the quantitative SRM / MRM assay of IRS1 protein in formalin-fixed tissue. Further analysis of the data from these experiments indicated that a preference for any specific peptide from any specific organ was not observed. Thus, each of these peptides is believed to be suitable for performing IRS1 protein SRM / MRM assays in a Liquid Tissue ™ lysate of any formalin-fixed tissue originating from any biological sample or any organ in the body.

The peptides in Table 1, or any combination of those peptides (e.g., one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more more of those peptides listed in Table 1, and in particular combinations with the peptides also found in Table 2) can be assayed by methods that do not depend on mass spectroscopy, including, but not limited to, immunological methods (p eg, Western blot or ELISA). Regardless of how the information on the amount (absolute or relative) of the peptide (s) is obtained, the information can be used in any of the methods described herein, including indicating (diagnosing) the presence of a cancer in a subject, determining the stage / grade / status of the cancer, providing a prognosis, or determining the therapeutic or treatment regimen for a subject / patient.

The present disclosure includes compositions comprising one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of the peptides of Table 1. In certain embodiments , the compositions comprise one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of the peptides in Table 2. Compositions comprising the peptides can include one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more peptides that are isotopically labeled. Each of the peptides can be labeled with one or more isotopes^{1 1 1} 4 x 4 7 ΊΑ 4 c 4 Q q ¹ independently selected from the group consisting of: O, O, S, N, C, H, or combinations thereof. Compositions comprising peptides of the IRS1 protein, whether or not isotope-labeled, need not contain all of the peptides of that protein (eg, a full set of tryptic peptides).

ES 2 642 807 T3

Optionally, the compositions do not contain one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more IRS1 peptides, and in particular the peptides that appear in Table 1 or Table 2. Compositions comprising the peptides may be in the form of dry or lyophilized materials, liquid (eg, aqueous) solutions or suspensions, matrices, or blots.

An important consideration in conducting an SRM / MRM assay is the type of instrument that can be used to analyze peptides. Although SRM / MRM assays can be developed and carried out on any type of mass spectrometer, including a MALDI, ion trap, or triple quadrupole, it is often considered the most advantageous instrumental platform for an SRM assay. / MRM is a triple quadrupole instrument platform. This type of mass spectrometer can be considered the most suitable instrument for analyzing a single target peptide isolated from a highly complex protein lysate that can consist of hundreds of thousands to millions of individual peptides from all the proteins contained in a cell.

To more efficiently implement an SRM / MRM assay for each peptide derived from the IRS1 protein, it is desirable to use information in addition to the peptide sequence in the analysis. That additional information can be used to direct and instruct the mass spectrometer (eg. a triple quadrupole mass spectrometer) to carry out the correct and focused analysis of the targeted peptide (s), so that the assay can be carried out efficiently.

Additional information on targeted peptides in general, and on specific IRS1 peptides, may include one or more of the peptide monoisotopic mass, precursor charge state, precursor m / z value, m / z of the transition ions, and the ion type of each transition ion. Additional peptide information that can be used to develop an SRM / MRM assay for IRS protein is shown for example for twelve (12) of the IRS1 peptides listed in Table 1, and is shown in Table 2 Similar additional information described for these twelve (12) IRS1 peptides shown, for example, in Table 2, can be prepared, obtained, and applied for the analysis of the other peptides contained in Table 1.

The method described below was used to: 1) identify candidate IRS1 protein peptides that can be used for a mass spectrometry-based SRM / MRM assay for IRS1 protein, 2) develop an assay, or assays, of Individual SRM / MRM to target IRS1 protein peptides for correlation and 3) apply quantitative assays for cancer diagnosis and / or optimal therapy choice.

Testing method

1. Identification of candidate SRM / MRM peptide fragments for the IRS protein

to. Prepare a Liquid Tissue ™ protein lysate from a formalin-fixed biological sample using a protease or proteases (which may or may not include trypsin), to digest the proteins

b. Analyze all protein fragments in the Liquid Tissue ™ lysate on an ion trap tandem mass spectrometer and identify all peptide fragments of the IRS1 protein, in which the individual peptide fragments do not contain any peptide modifications such as phosphorylations or glycosylations

c. Analyze all protein fragments in the Liquid Tissue ™ lysate on an ion trap tandem mass spectrometer and identify all peptide fragments of the IRS1 protein that carry peptide modifications such as, for example, phosphorylated or glycosylated residues

d. All peptides generated can potentially be measured by a specific digestion method of the intact, full-length IRS1 protein, but the preferred peptides used for SRM / MRM assay development are those that are identified by mass spectrometry directly in a Liquid Tissue ™ complex protein lysate prepared from a formalin-fixed biological sample

and. Peptides that are specifically modified (phosphorylated, glycosylated, etc.) in patient tissue and that are ionized, and thus detected, on a mass spectrometer when a Liquid Tissue ™ lysate of a biological sample is analyzed fixed in formalin are identified as candidate peptides to test for peptide modifications of the IRS1 protein

two. Mass Spectrometry Assay for Peptide Fragments of the IRS1 Protein

to. The SRM / MRM assay on a triple quadrupole mass spectrometer for individual peptide fragments identified in a Liquid Tissue ™ lysate is applied to IRS protein peptides

i. Determine the optimal retention time for a peptide fragment for optimal chromatography conditions including, but not limited to, gel electrophoresis, liquid chromatography,

ES 2 642 807 T3 capillary electrophoresis, nano-reverse phase liquid chromatography, high performance liquid chromatography, or reverse phase high performance liquid chromatography ii. Determine the monoisotopic mass of the peptide, the state of charge of the precursor for each peptide, the m / z value of the precursor for each peptide, the m / z of the transition ion for each peptide, and the ion type of each transition ion. for each peptide fragment, to develop an SRM / MRM assay for each peptide.

iii. The SRM / MRM assay can then be carried out by using the information from (i) and (ii) on a triple quadrupole mass spectrometer, in which each peptide has a distinctive SRM / MRM peak and Unique that exactly defines the unique SRM / MRM assay as performed on a triple quadrupole mass spectrometer

b. Carry out the SRM / MRM analysis so that the amount of the peptide fragment of the IRS1 protein that is detected, as a function of the area of ​​the unique SRM / MRM distinctive peak from an SRM / mRm mass spectrometric analysis , can indicate both the relative and the absolute amount of the protein in a particular protein lysate.

i. Relative quantification can be achieved:

1. Determining the increased or decreased presence of IRS1 protein by comparing the SRM / MRM signature peak area of ​​a particular IRS1 peptide detected in a Liquid Tissue ™ lysate from a formalin-fixed biological sample to the same signature peak area of SRM / MRM of the same IRS1 peptide fragment in at least one second, third, fourth, or more Liquid Tissue ™ lysates from at least one second, third, fourth, or more formalin-fixed biological samples

two. Determining the increased or decreased presence of IRS protein by comparing the sRm / MRM signature peak area of ​​a particular IRS1 peptide detected in a Liquid Tissue ™ lysate from a formalin-fixed biological sample to the signature SRM peak areas / MRM developed from peptide fragments of other proteins, in other samples obtained from different and separate biological sources, wherein the comparison of the distinctive SRM / MRM peak areas between the 2 samples for a peptide fragment are normalized to the amount of protein analyzed in each sample.

3. Determining the increased or decreased presence of the IRS protein by comparing the SRM / MRM signature peak area for a particular IRS1 peptide to the SRM / MRM signature peak areas of other peptide fragments derived from different proteins in the same lysate of Liquid Tissue ™ from the formalin-fixed biological sample to normalize the varying levels of IRS1 protein to the levels of other proteins that do not vary their expression levels in various cellular conditions.

Four. These assays can be applied to both unmodified peptide fragments and modified peptide fragments of the IRS1 protein, in which the modifications include, but are not limited to, phosphorylation and / or glycosylation, and in which the relative levels of the modified peptides are they are determined in the same way that the relative amounts of the unmodified peptides are determined.

ii. Absolute quantification of a particular peptide can be achieved by comparing the SRM / MRM signature peak area for a particular IRS1 protein peptide fragment in a single biological sample to the SRM / MRM signature peak area of ​​an internal fragment standard. peptide added to the protein lysate of the biological sample

1. The internal standard is a labeled synthetic version of the peptide fragment of the IRS1 protein under investigation. This standard is added to a sample in known amounts, and the area of ​​the distinctive SRM / MRM peak can be determined for the internal peptide fragment standard and the native peptide fragment in the biological sample separately, followed by comparison of the areas. of both peaks

two. This can be applied to unmodified peptide fragments and modified peptide fragments, where the modifications include, but are not limited to, phosphorylation and / or glycosylation, and where the absolute levels of the modified peptides can be determined in the same manner as The absolute levels of the unmodified peptides are determined.

3. Application of the Quantification of Peptide Fragments to the Diagnosis and Treatment of Cancer

to. Carry out a relative and / or absolute quantification of the levels of peptide fragments of the IRS1 protein and demonstrate that the previously determined association is confirmed, as understood