Abstract:
An integrated proteomics sample preparation device and method for in-gel digestion of proteins and for desalting and concentrating samples prior to further analysis such as by MALDI TOF and/or electro-spray ionization (ESI) mass spectrometry. The device in accordance with an embodiment of the present invention includes a plurality of wells in fluid communication with a an outlet or drainage opening containing a three dimensional structure comprising a plurality of sorptive particles entrapped in a porous polymer matrix so as to form a device capable of carrying out solid phase extraction. In a preferred embodiment, the wells are configured so as to prevent a sample carrier present in the wells from clogging the outlet when subjected to a driving force such as vacuum. The device also reduces or eliminates overflowing of a well in the event a drain becomes clogged during automated operation.

Description:
This application is a continuation of U.S. patent application Ser. No. 10/154,550 filed May 24, 2002, the disclosure of which is incorporated herein by reference 
    
    
     BACKGROUND OF THE INVENTION 
     Matrix-assisted laser desorption/ionization (MALDI) analysis is a useful tool for solving structural problems in biochemistry, immunology, genetics and biology. Samples are ionized and a time of flight (TOF) analyzer is used to measure ion masses. TOF analysis begins when ions are formed and are accelerated to a constant kinetic energy as they enter a drift region. 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 proteome research. Current trends in research are to analyze larger and larger numbers of samples using automated handling equipment or robotics. Quantities of individual samples are from the nano-mole levels to femto-mole levels. As a result, instrumentation is becoming more sensitive and a need exists for sample handling formats to be miniaturized, high density and disposable. 
     In-gel digestion of protein is a proteomics method that has many sample preparation steps prior to sample analysis (such as by MALDI TOF MS). Briefly, upon separation in the electrophoresis gel, the proteins in a sample are stained for detection and portions of the gel containing the protein of interest are excised. 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 out of the gel portion into solution. After purification of the peptides, analysis of the sample is carried out. 
     Simultaneous preparation and analysis of multiple samples is often desirable. Multiwell plates have been developed for simultaneous assay, typically consisting of 96, 384 or 1536 reaction vessels or wells per plate. It would be desirable to use multiwell plates also for sample handling and preparation, such as the removal of undesired salts and biochemical substances to improve the resolution and selectivity of the mass spectrum. 
     In this connection, EP 1 151 793 discloses a microtiter plate having lyophobic porous bottoms. Gel pieces containing proteins are placed in the wells of the plate and digested with 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 process. In addition, it is not easily adaptable to automation or robotics. It would be highly desirable to use the microtiter plate format for enzyme digestion and protein capture that does not require centrifugation, and that is readily adaptable to automation. 
     Another difficulty is that the gel plugs are deformable and have a similar diameter to the cone-shaped drain outlet of the plate. When vacuum filtered, the gel plugs clog the outlet, causing the well either to not drain or overflow with multiple solution additions, thus contaminating adjacent wells. 
     It is therefore an object of the present invention to provide a sample preparation method for desalting and purification of samples prior to matrix assisted laser desorption ionization time-of-flight (MALDI TOF) or electro-spray ionization (ESI) mass spectrometry or other analysis methods, that also can be used for digestion of protein, particularly in-gel digestion. 
     It is a still further object of the present invention to provide a high-density multi-well device wherein various arrays within the device contain chromatographic media having the same or different chemistries, and wherein in-gel digestion of protein is carried out using vacuum 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 robotics liquid handling equipment. 
     These and other objects will be made apparent by the following description. 
     SUMMARY OF THE INVENTION 
     The problems of the prior art have been overcome by the present invention, one embodiment of which provides an integrated proteomics sample preparation device and method for digestion of proteins and for desalting and concentrating samples prior to further analysis such as by MALDI TOF and/or electro-spray ionization (ESI) mass spectrometry. The device and method of the present invention allows for digestion, desalting and concentration of sample prior to MALDI TOF MS analysis. More specifically, the device in accordance with an embodiment of the present invention includes a plurality of wells each in fluid communication with a respective outlet or drainage opening, optionally containing a three dimensional structure comprising a plurality of sorptive particles entrapped in a porous polymer matrix so as to form a device capable of carrying out solid phase extraction. In a preferred embodiment, the wells are configured so as to prevent a sample carrier, such as a gel piece inserted in the wells from clogging the outlet when subjected to a driving force such as vacuum. The device also reduces or eliminates cross-contamination between wells in the event a drain becomes clogged. 
     The present invention is also directed towards a method of sample preparation using the device of the present invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of a single well for a multiwell sample preparation device in accordance with the present invention; 
         FIG. 2  is a perspective view of a single well for a multiwell sample preparation device shown containing a gel piece in accordance with the present invention; 
         FIG. 3  is a cross-sectional view of a single well for a multiwell sample preparation device shown containing a gel piece in accordance with the present invention; 
         FIG. 4  is a enlarged perspective view of a single well for a multiwell sample preparation device shown containing a gel piece in accordance with the present invention; 
         FIG. 5  is a cross-sectional view of a single well for a multiwell sample preparation device shown containing a gel piece (in phantom) and a matrix having adsorptive properties in the drain in accordance with the present invention; 
         FIG. 5A  is a top view of fluid passageways formed in the well in accordance with the present invention; 
         FIG. 6  is a perspective view of two side-by-side wells of a multi-well sample preparation device in accordance with the present invention; 
         FIG. 7  is a perspective view of a solid rendering of the drain and passageways of a well in accordance with the present invention; 
         FIG. 8  is a cross-sectional view of a single well for a multiwell sample preparation device shown containing a gel piece (in phantom) and a matrix having adsorptive properties in the drain in accordance with an alternative embodiment of the present invention; 
         FIG. 8A  is a top view of fluid passageways formed in the well in accordance with the embodiment of  FIG. 8 ; 
         FIG. 9  is a perspective view of a well having a dividing member in accordance with an embodiment of the present invention; and 
         FIG. 10  is a cross-sectional view of a well having raised bumps in accordance with an 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 particularly polypropylene, are preferred materials. 
     Turning now to  FIGS. 1 and 2 , there is shown generally at  10  a single well  12  suitable for use in a single well or a multiwell sample preparation device that has a plurality of wells. A well  12  is defined by a vertically extending fluid impervious side wall and a sloping bottom portion. The middle and upper portions of the 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  tapers downwardly, in the direction of fluid flow, towards a bottom portion  13 , which slopes inwardly towards a center, thereby having a frusto-conical configuration. Bottom portion  13  has a drain  15  that is preferably centrally located in the well  10 . 
     Formed in the bottom portion  13  of the well  10  are one or more fluid passageways  18 . The fluid passageway(s)  18  modify the otherwise relatively smooth or even surface of bottom portion  13  and effectively provide a gap or space between a sample carrier  20 , such as a gel piece ( FIG. 2 ), that is contained in well  12  and supported by the bottom portion  13 , and the drain  15 . The sample carrier can be a liquid but is preferably a solid, such as a gel, coated bead or a membrane. In order to insure fluid flow between the well  12  and drain  15  when the carrier  20  is present in the well  12 , the smallest dimension of each passageway  18  should be less than the smallest dimension of the carrier  20 , so that the carrier  20  cannot be positioned in the passageway  18  to block fluid flow into the drain  15 . In this way, at least a portion of the fluid passageway(s)  18  is always in fluid communication with the drain  15  and cannot be blocked or clogged by a carrier  20  when placed in the well  12 , as exemplified by illustration in  FIGS. 2 ,  3  and  4 . Where the carrier is a gel piece, it is noted that typically circular plug cutters found in automated picker robots cut the gel portion uniformly. However, the present invention is not limited to uniformly-shaped carriers, as the fluid passageway(s)  18  are configured to prevent fluid blockage even when carriers of irregular shape 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 insure fluid flow around the sample carrier, preferably there is a plurality of such passageways. At least two passageways  18 , most preferably three passageways  18 , formed symmetrically about the drain  15  as best seen in  FIGS. 5A and 7 , is the particularly preferred arrangement. The symmetrical arrangement of the passageways about the drain  15  ensure that regardless of the orientation of the carrier  20  in the well  12 , fluid communication between the well  12  and the drain  15  will be maintained. The shape and topology of the passageway(s)  18  are not particularly limited, as long as they do not match that of the carrier  20 . Preferably the passageway(s)  18  are lobes, but a square, stepped round, cone with a bump or cross bar also are suitable configurations. As best seen in  FIG. 7 , the lobes preferably taper so that they are deeper as they approach the drain  15 . 
     The passageway(s)  18  are preferably formed by creating asymmetry in the surface of the bottom portion  13 . This can be accomplished by providing grooves in the surface, or by providing raised portions or protrusions in or on the surface such as a cross bar  117  ( FIG. 9 ) or ribs or bumps  118  ( FIG. 10 ). Preferably the passageways  18  are grooves having a depth of about 0.2 mm, a width of about 0.25 mm and a length of about 1 mm. In the embodiment utilizing protrusions, the protrusions are designed so 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 situated over the drain  15  in such as way as to block fluid flow to the drain  15 . 
     As seen in  FIGS. 1 and 7 , the drain  15  is a bore, preferably cylindrical and axially aligned with the central longitudinal axis of the well  12 . The drain  15  is in fluid communication with the passageways  18 . At least a portion of the drain  15  preferably includes an adsorptive composite structure  25  ( FIGS. 5 and 5A ). Suitable adsorptive composite structures are cast-in-place polymer bound, particle laden adsorptive membrane structures, such as those comprised of chromatographic beads which have been adhered together with a binder and disclosed in U.S. Pat. No. 6,048,457, the disclosure of which is hereby incorporated by reference. One such preferred structure is a three-dimensional structure comprising a plurality of sorptive particles entrapped in a porous polymer matrix and having an aspect ratio (average diameter to average thickness) of less than about 10, preferably less than about 5. The structure  25  is preferably coterminous with the bottom of the drain  15 , and extends into the drain  15 , preferably extending through the entire depth of the drain  15  and may extend into the passageway(s)  18  as shown in  FIG. 5 . Although the composite structure  25  can 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 the passageway(s)  18  is not blocked by the carrier  20 . 
     As shown in  FIGS. 8 and 8A , the composite structure can be formed to have one or more dimensions that are greater than the largest dimension of the carrier  20 , and thus ensure fluid communication between the well and the drain without the formation of a passageway to maintain surface area for flow. For example, the face shape of the composite structure can be a circle having a long leg  25 A extending from the circle, or can be in the shape of an eye, thereby ensuring that some surface of the composite structure remains unobstructed and available for flow regardless of the orientation of the carrier  20 . 
     Devices in accordance with the present invention may incorporate a plurality of composite structures having resin materials with different functional groups to fractionate analytes that vary by charge, size, affinity and/or hydrophobicity; alternately, a plurality of devices containing different individual functional membranes may be used in combination to achieve a similar result. Similarly, one or more membranes can be cast in a suitable housing and functionality can be added before or after casting. 
     In an alternative embodiment, the drain can be devoid of any media, and the device used as a non-clogging processing device that delivers digested proteins to a collection well for analysis or concentration, for example. 
     After the proteins in the carrier are stained and small pieces of the carrier containing the protein(s) of interest are excised from the site of the stain, 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. Sufficient enzyme is added to effectively digest the protein(s). Preferably an excess of enzyme is added, and in sufficient amount to submerge the carrier in each well. After an incubation period to allow the protein digestion to take place and the resulting peptides to diffuse out of the carrier, vacuum is applied to each well, preferably to create a pressure differential of about 5-10 psi, to cause extracted peptides to flow into the drain  15  where they are adsorbed (when media is present) and can then be washed in the conventional manner and freed from buffers, salts and other contaminants. Concentrated peptides then can be eluted and delivered to a suitable target or presentation device for analysis such as by MALDI TOF MS. 
     During an automated multi-addition procedure, there is the possibility that wells can overflow, if blocked. The present invention reduces or eliminates the possibility of contamination of other wells as a result of the overflow by incorporating an overflow control feature into the device of the present invention. Specifically, with reference to  FIG. 6 , surrounding at least a portion of each well  12  is a recess  30 . The recess preferably is formed from the top surface  29  of each well  12 , which generally corresponds to the top surface  32  of the tray  35 , and extends downward (towards the drain  15 ) about 50% of the length of the well  12 , where it terminates in bottom wall  34 . The depth of the recess is not critical, as long as is sufficient to contain the overflow volume from at least one well.