Anti-clogging device and method for in-gel digestion applications

ES 2 389 463 T3

DESCRIPTION

Anti-clogging device and procedure for gel digestion applications

Matrix Assisted Laser Ionization / Desorption Analysis (MALDI) is a useful tool for solving structural problems and biochemistry, immunology, genetics and biology. The samples are ionized and the time of flight (TOF) analyzer is used to measure ion masses. TOF analysis begins when ions are formed and accelerated to constant kinetic energy as they enter a region of flow. They arrive at a detector following flight times that are proportional to the square root of their masses. A mass spectrum is created because ions of different mass arrive at the detector at different times.

Mass spectrometry can be a particularly powerful tool in the fields of drug discovery and development, genotyping, and proteasome research. Current trends in research are to analyze increasing numbers of samples using automated handling equipment or robots. The individual sample amounts are derived from nanomole levels to femtomol levels. As a result, instrumentation is increasingly sensitive and there is a need for miniaturized, high-density, disposable sample handling formats.

Protein gel digestion is a proteomic procedure that has many steps of sample preparation prior to sample analysis (such as MALDI-TOF MS). Briefly, after separation on the electrophoresis gel, the proteins in a sample are stained for detection and the portions of the gel containing the protein of interest are cut out. The stain is then removed from these gel portions and an enzyme solution is used to selectively digest the protein sample to form peptides that migrate from the gel portion into solution. After the purification of the peptides, the analysis of the sample is carried out.

Simultaneous preparation and analysis of multiple samples is often desirable. Multiple well plates have been developed for simultaneous assays, typically consisting of 96, 384, or 1536 reaction vessels or wells per plate. It would be desirable to use multi-well plates also for sample handling and preparation, such as removal of unwanted salts and biochemicals to improve resolution and mass spectrum selectivity.

In this regard, EP 1 151 793 discloses a microtiter plate having lyophobic porous bottoms. Protein-containing gel pieces are placed in the wells of the plate and digested with the enzyme. The enzyme is then removed from the gel pieces by centrifugation and applied to a MALDI sample carrier plate for analysis.

However, using centrifugation to bind, wash and elute is a time consuming procedure. Furthermore, it is not easily adaptable to automation or robotics. It would be highly desirable to use the microtiter plate format for enzymatic digestion and protein capture that does not require centrifugation and is easily adaptable to automation.

Another difficulty is that the gel plugs are deformable and have a diameter similar to the cone-shaped drain outlet of the plate. When vacuum filtered, gel plugs clog the outlet and cause the well to not drain or to overflow with multiple additions of solution, thereby contaminating adjacent wells,

It is therefore an object of the present invention to provide a sample preparation method for desalting and purifying samples prior to matrix-assisted laser ionization-desorption with time of flight (MALDITOF) or electrospray ionization mass spectrometry (ESI ) or other analytical procedures, which can also be used for protein digestion, in particular gel digestion.

It is yet another object of the present invention to provide a high density multiwell device in which various arrays within the device contain chromatographic media having the same or different chemistries and in which protein gel digestion is carried out using emptiness as a driving force.

It is a further object of the present invention to provide a sample preparation system and method that is suitable for automated robotic liquid handling equipment.

These and other objects will be apparent from the following description.

Summary of the Invention

The present invention has overcome the problems of the prior art, the essential and optional features of which are set forth in the accompanying main claims and subclaims respectively. One embodiment of the invention provides an integrated proteomic sample preparation device and procedure for protein digestion and for desalting and concentrating samples prior to subsequent analysis, such as by MALDI TOF and / or electrospray ionization mass spectrometry ( ESI). The device and method of the present invention allows for digestion, desalting, and concentration of samples prior to MALDI TOF MS analysis. More specifically, the device according to one embodiment

ES 2 389 463 T3 of the present invention includes a plurality of wells, each in fluid communication with a respective outlet or drain opening, optionally containing a three-dimensional structure comprising a plurality of portion particles entrapped in a porous polymeric matrix to form a device capable of carrying out solid phase extraction. In a preferred embodiment, the wells are configured in such a way as to prevent a sample carrier, such as a piece of gel inserted into the wells, from clogging the outlet when subjected to a driving force such as a vacuum. The device also reduces or eliminates cross-contamination between wells in the event that a drain becomes clogged.

The present invention is also directed to a sample preparation method using the device of the present invention.

Brief description of the figures

Figure 1 is a perspective view of a single well for a multi-well sample preparation device in accordance with the present invention.

Figure 2 is a perspective view of a single well for a multi-well sample preparation device containing a piece of gel in accordance with the present invention.

Figure 3 is a cross-sectional view of a single well for a multi-well sample preparation device containing a piece of gel in accordance with the present invention.

Figure 4 is an enlarged perspective view of a single well for a multi-well sample preparation device containing a piece of gel in accordance with the present invention.

Figure 5 is a cross-sectional view of a single well for a multi-well sample preparation device containing a gel piece (phantom) and a matrix having drain adsorption properties in accordance with the present invention. .

Figure 5A is a top view of fluid passageways formed in a well in accordance with the present invention.

Figure 6 is a perspective view of two adjacent wells of a multi-well sample preparation device in accordance with the present invention.

Figure 7 is a perspective view of a solid exiting the drain and passageways in accordance with the present invention.

Figure 8 is a cross-sectional view of a single well for a multi-well sample preparation device containing a gel piece (phantom) and a matrix having drain adsorption properties in accordance with an alternate embodiment. of the present invention.

Figure 8A is a top view of fluid passageways formed in the well in accordance with the embodiment of Figure 8.

Figure 9 is a perspective view of a well having a dividing member in accordance with one embodiment of the present invention.

Figure 10 is a cross-sectional view of a well having protrusions in accordance with one embodiment of the present invention.

Detailed description of the invention

Suitable substrate materials for the sample preparation device of the present invention are not particularly limited and include plastics (such as polyethylene and polypropylene), glass, and stainless steel. The substrate materials should not interfere with the operation of the device or the chemicals to be used in the procedure. Polyolefins, and in particular polypropylene, are preferred materials.

Turning now to Figures 1 and 2, a single well 12 suitable for use in a single-well or multi-well sample preparation device having a plurality of wells is shown generally at 10. A well 12 is defined by a vertically extending fluid impervious side wall and a sloping lower portion. The middle and upper portions of well 12 preferably have a uniform diameter and are substantially cylindrical in cross section, although other configurations are contemplated and within the scope of the present invention. The lower portion of the well 12 gradually tapers downward, in the direction of fluid flow, toward the lower portion 12, which slopes inwardly toward a center, thereby having a frusto-conical configuration. The lower portion 13 has a drain 15 which is located, preferably, centrally in the well 10.

Formed in the lower portion 13 of the well 10 are one or more fluid passageways 18. The fluid passage (s) 18 modify the otherwise relatively smooth or uniform surface of the lower portion 13 and provide

ES 2 389 463 T3 effectively provides a gap or space between a sample carrier 20, such as a piece of gel (Figure 2), which is contained in well 12 and supported by lower portion 13, and drain 15. The sample carrier is a solid, such as a gel, a coated bead, or a membrane. In order to ensure fluid flow between well 12 and drain 15 when conveyor 20 is present in well 12, the smallest dimension of each passageway 18 should be less than the smallest dimension of conveyor 20, so that the conveyor 20 cannot be placed in the passageway 18 to block the flow of fluid to the drain 15. Thus, at least a portion of the passageway (s) 18 is always in fluid communication with drain 15 and cannot be blocked or obstructed by a conveyor 20 when placed in well 12, as stated exemplified by illustration in Figures 2, 3 and 4. When the conveyor is a piece of gel, it is observed that normally circular plug cutters in collecting robots cut the gel portion evenly. However, the present invention is not limited to uniformly shaped conveyors, as the fluid passage (s) 18 are configured to prevent fluid blockage even when the irregularly shaped conveyors are present in the well. 12. For example, a single slit that is longer than the carrier is within the scope of the present invention.

Although a single passageway 18 is sufficient to ensure fluid flow around the sample carrier, there are preferably a plurality of such passageways. At least two passageways 18, more preferably three passageways 18, formed symmetrically around drain 15 as best seen in Figures 5A and 7, is the particularly preferred arrangement. The symmetrical arrangement of the passageways around drain 15 ensures that, regardless of the orientation of conveyor 20 in well 12, fluid communication between well 12 and drain 15 will be maintained. The shape and topology of the passageway (s) 18 are not particularly limited, as long as they do not coincide with that of the conveyor 20. Preferably, the passageway (s) 18 are lobar, but a square, rounded corners, cone with a bulge, or a bar through are suitable configurations. As best seen in Figure 7, the lobes taper so that they are deeper as they approach drain 15.

The passageway (s) 18 are preferably formed by creating asymmetry in the surface of the lower portion 13. This can be achieved by providing grooves in the surface and by providing raised portions or protrusions in or on the surface, such as a cross bar (Figure 9) or ribs or protrusions 118 (Figure 10), Preferably, the passageways 18 are grooves that they have a depth of about 0.2 mm, a width of about 0.25 mm, and a length of about 1 mm.

In the embodiment using protrusions, the protrusions are designed such that the largest opening in the drain is smaller than the smallest dimension of the sample carrier. The objective is to prevent the sample carrier 20 from being positioned over the drain 15 in such a way as to block the flow of fluid to the drain 15.

As seen in Figures 1 and 7, drain 15 is a hole, preferably cylindrical and axially aligned with the central longitudinal axis of well 12. Drain 15 is in fluid communication with passageways 18. At least a portion of the Drain 15 preferably includes a composite adsorption structure 25 (Figures 5 and 5A). Suitable adsorption composite structures are molded polymer-bound particle loaded adsorption membrane structures, such as those composed of chromatographic beads that have been adhered together with a binder and disclosed in US Patent No. 6,048,457, which disclosure is incorporated herein by reference. One of these preferred structures is a three-dimensional structure comprising a plurality of portion particles entrapped in a porous polymeric matrix and having a proportionality ratio (ratio of mean diameter to mean thickness) of less than about 10, preferably less than about 5. Structure 25 is preferably adjacent to the bottom of drain 15 and extends into drain 15, preferably extends through the entire depth of drain 15 and may extend into passageway (s) 18 as shown in Figure 5. Although the composite structure 25 may also completely fill the passageway (s) 18, it is preferred that a portion (preferably the upper half), such as 50% of the passageway (s) 18 remains devoid of structure 25 to ensure that passageway (s) 18 is (are) not blocked by conveyor 20.

As shown in Figures 8 and 8A, the composite structure can be formed to have one or more dimensions that are larger than the largest dimension of the conveyor 20 and thus ensure fluid communication between the well and the drain. without the formation of a passageway to maintain the surface area for flow. For example, the front shape of the composite structure may be a circle having a long limb 25A extending from the circle or it may be in the shape of an eye, ensuring that some surface of the composite structure remains unobstructed. and available for flow, regardless of the orientation of the conveyor 20.

Devices according to the present invention can incorporate a plurality of composite structures having resin materials with different functional groups to fractionate analytes that vary in charge, size, affinity and / or hydrophobicity; alternatively, a plurality of devices containing different individual functional membranes can be used in combination to achieve a similar result. Similarly, one or more membranes can be poured into a suitable housing and the functionality can be added before or after pouring.

ES 2 389 463 T3

In an alternative embodiment, the drain can be devoid of any media and the device can be used as a non-clog processing device that releases the digested proteins to a collection well for analysis or concentration, for example.

After the proteins and carrier are stained and the small pieces of carrier containing the protein (s) of interest are excised from the staining site, each carrier piece is placed in a respective well. A suitable amount of proteolytic enzyme solution is added to each well, such as by pipetting. Enough enzyme is added to effectively digest the protein (s). Preferably an excess of enzyme is added and in an amount sufficient to immerse the carrier in each well. After an incubation period to allow protein digestion and the resulting peptides to diffuse out of the carrier, a vacuum is applied to each well, preferably to create a differential pressure of approximately 5-10 psi, to cause the extracted peptides to flow into the carrier. drain 15 where they are adsorbed (when media is present) and then can be washed in a conventional manner and freed of buffers, salts and other contaminants. The concentrated peptides can be eluted and delivered to a suitable target or display device for analysis, such as by MALDI TOF MS.

During an automated multi-add procedure, there is a possibility that the wells could overflow if they become blocked. The present invention reduces or eliminates the possibility of contamination of other wells as a result of overflow by incorporating an overflow control feature in the device of the present invention. Specifically, referring to Figure 6, surrounding at least a portion of each well 12 is a burr 30. The flash is preferably formed from the upper surface 29 of each well 12, which generally corresponds to the upper surface 32 of the tray 35 and extends downward (towards the drain 15) approximately 50% of the length of the well 12, where it ends at bottom wall 34. The depth of the burr is not critical, as long as it is sufficient to contain the overflow volume of at least one well.