Probe for mass spectrometry

Abstract

The present invention relates to a probe for the analysis of one or more analytes, particularly proteins or compounds capable of binding or otherwise interacting therewith, by laser desorption/ionisation mass spectrometry, more particularly MALDI MS. It also relates to a protein microarray, a method of producing a protein microarray and a method of analysing a protein microarray. The probe comprises a support having an electroconductive target surface thereon characterized in that the target surface comprises a micro array having a plurality of discrete target areas presenting one or more analyte capture moieties. Each discrete target area has an area of less than 1000 μm 2 , more preferably still less than 500 μm 2 , and more preferably still less than 100 μm 2 .

1 - 73 . (canceled)  
     
     
         74 . A probe for use with a laser desorption/ionisation mass spectrometer, which probe comprises a support having an electroconductive target surface thereon, said target surface comprising a layer that is resistant to non-specific protein binding, said layer incorporating protein repellent molecules and one or more high affinity analyte capture moieties; wherein said high affinity analyte capture moieties are incorporated homogeneously in said layer in small proportions relative to the protein repellent molecules.  
     
     
         75 . A probe as claimed in  claim 74 , wherein the proportion of said high affinity analyte capture moieties relative to said protein repellent molecules in said layer is less than 20%.  
     
     
         76 . A probe as claimed in  claim 74  or  claim 75 , wherein the proportion of said high affinity analyte capture moieties relative to said protein repellent molecules in said layer is in the range 1-20%.  
     
     
         77 . A probe as claimed in  claim 76 , wherein the proportion of said high affinity analyte capture moieties relative to said protein repellent molecules in said layer is 1%, 2%, 10% or 20%.  
     
     
         78 . A probe as claimed in  claim 74 , wherein said high affinity analyte capturing moieties are homogeneously disposed across substantially the whole of said surface.  
     
     
         79 . A probe as claimed in  claim 74 , wherein said target surface comprises a microarray having a plurality of discrete target areas, and said high affinity analyte capturing moieties are homogenously disposed in discrete target areas.  
     
     
         80 . A probe as claimed in  claim 79 , wherein said high affinity analyte capture moieties are homogenously disposed only in said discrete target areas.  
     
     
         81 . A probe as claimed in  claim 79 , wherein said high affinity analyte capture moieties are disposed in the discrete target areas in a defined orientation.  
     
     
         82 . A probe as claimed in  claim 79 , wherein the surface in the discrete target areas is substantially planar.  
     
     
         83 . A probe as claimed in  claim 80 , wherein the discrete target areas are flat bottomed wells.  
     
     
         84 . A probe as claimed in  claim 74  which probe can bind an analyte at a concentration of below 10 15  molecules per 1000 μm 2 , more preferably at a concentration of below 10 12  molecules per 1000 μm 2 , more preferably at a concentration of below 10 9  molecules per 1000 μm 2 , more preferably still at a concentration of below 10 6  molecules per 1000 μm 2 .  
     
     
         85 . A probe as claimed in  claim 74 , wherein said high affinity analyte capture moieties are protein capture moieties.  
     
     
         86 . A probe as claimed in  claim 85 , wherein said high affinity protein capture moieties are not an antibody.  
     
     
         87 . A probe as claimed in  claim 74 , wherein said high affinity analyte capture moieties are small molecules.  
     
     
         88 . A probe as claimed in  claim 87 , wherein said small molecules are less than 2 kDa, preferably less than 1 kDa, more preferably less than 500 Da.  
     
     
         89 . A probe as claimed in  claim 74 , wherein the binding affinity (Kd) between said high affinity analyte capture moieties and their binding partners is at least 10 −7 M, more preferably at least 10 −   9  M, more preferably at least 10 −12 M and more preferably still at least 10 −15 M.  
     
     
         90 . A probe as claimed in  claim 74 , wherein the support is a glass slide, or a MALDI target.  
     
     
         91 . A probe as claimed in  claim 74 , wherein said electroconductive surface comprises a metal or a semi conductor.  
     
     
         92 . A probe as claimed in  claim 91 , wherein said metal is selected from gold, silver, platinum, iridium, iron, nickel, cobalt, copper or a mixture or alloy thereof, and the semi conductor is selected from silicon, graphite or germanium.  
     
     
         93 . A probe as claimed in  claim 74 , wherein said high affinity analyte capture moiety is attached directly to the electroconductive target surface.  
     
     
         94 . A probe as claimed in  claim 74 , wherein said high affinity analyte capture moiety is indirectly attached to the electroconductive target surface.  
     
     
         95 . A probe as claimed in  claim 94 , wherein said high affinity analyte capture moiety is attached via one or more linker molecules.  
     
     
         96 . A probe as claimed in  claim 95 , wherein said linker molecules comprise a poly amino acid or an alkane thiol.  
     
     
         97 . A probe as claimed in  claim 96 , wherein said poly amino acid is poly-L-lysine, poly-L-aspartic acid, poly-L-glutamic acid or mixtures of any other known amino acids with the three aforementioned amino acids.  
     
     
         98 . A probe as claimed in  claim 85 , wherein at least one high affinity protein capturing moiety binds biotin or a bleomycin resistance protein.  
     
     
         99 . A probe as claimed in  claim 98 , wherein said high affinity protein capturing moiety is streptavidin, avidin, neutravidin or bleomycin.  
     
     
         100 . A probe as claimed in  claim 74 , wherein said layer comprises a polymer or a self assembled monolayer (SAM) which is responsible for the generally protein repellent nature of the layer.  
     
     
         101 . A probe as claimed in  claim 100 , wherein the polymer comprises polyethylene glycol (PEG), dextran, polyurethane or polyacrylamide.  
     
     
         102 . A probe as claimed in  claim 100 , wherein the polymer is bound to the probe surface via one or more linker molecules.  
     
     
         103 . A probe as claimed in  claim 102 , wherein said high affinity analyte capturing moiety is attached to the surface via the polymer and/or the linker molecules.  
     
     
         104 . A probe as claimed in  claim 74 , wherein a single common high affinity analyte capture moiety is provided on the surface.  
     
     
         105 . A probe as claimed in  claim 74 , wherein a plurality of different high affinity analyte capturing moieties are provided on the surface.  
     
     
         106 . A probe as claimed in  claim 74 , further comprising a captured analyte, which analyte is captured in a plurality of discrete target areas as a microarray.  
     
     
         107 . A probe as claimed in  claim 106 , wherein said discrete target areas are arranged in a spatially defined manner.  
     
     
         108 . A probe as claimed in  claim 106 , wherein each discrete target area has an area of less than 1000 μm 2 , more preferably still less than 500 μm 2 , and more preferably still less than 100 μm 2 .  
     
     
         109 . A probe as claimed in  claim 106 , wherein each discrete target area has an area of less than 785 μm 2  more preferably less than 392 μm 2  more preferably still less than 78 μm 2 .  
     
     
         110 . A probe as claimed in  claim 106 , wherein said discrete target areas are substantially circular.  
     
     
         111 . A probe as claimed in  claim 106 , wherein said discrete target areas are arranged in matrices.  
     
     
         112 . A probe as claimed in  claim 111 , wherein there are a plurality of matrices on the target surface.  
     
     
         113 . A probe as claimed in  claim 111 , wherein the matrices comprise at least two rows and two columns of discrete target areas.  
     
     
         114 . A probe as claimed in  claim 106  comprising at least ten, more preferably at least one hundred, more preferably still at least one thousand, and more preferably still at least ten thousand discrete target areas.  
     
     
         115 . A probe as claimed in  claim 106 , wherein there is a spacing between adjacent discrete target areas in a matrix of less than 1 mm.  
     
     
         116 . A probe as claimed in  claim 106 , wherein said captured analyte comprises a protein.  
     
     
         117 . A probe as claimed in  claim 116 , wherein the protein is a fusion protein.  
     
     
         118 . A probe as claimed in  claim 117 , wherein said fusion protein comprises a biotin carboxyl carrier protein (BCCP).  
     
     
         119 . A probe as claimed in  claim 116 , wherein the fusion protein comprises a phleomycin/zeocin resistance protein.  
     
     
         120 . A probe as claimed in  claim 106 , wherein said captured analyte has a further molecule bound to it.  
     
     
         121 . A probe as claimed in  claim 120 , wherein said further molecule is a small molecule, a protein or a nucleic acid.  
     
     
         122 . A probe as claimed in  claim 106 , wherein said analyte is printed onto the surface.  
     
     
         123 . A probe as claimed in  claim 122 , wherein said analyte is printed using inkjet printing, piezo electric printing or contact printing.  
     
     
         124 . A probe as claimed in  claim 123 , wherein for contact printing said analyte is applied using a split pin, solid pin or a hollow pin.  
     
     
         125 . A method of producing a protein microarray for use with a laser desorption ionisation/mass spectrometer, said method comprising depositing protein on the electroconductive target surface of a probe as claimed in  claim 85  such that said protein is deposited in discrete target areas as a protein microarray.  
     
     
         126 . A method of analysing a protein microarray by laser desorption/ionisation mass spectrometry, said method comprising depositing protein on an electroconductive target surface of a probe, which probe comprises a support having said target surface thereon, said surface presenting one or more protein capture moieties, such that said protein is deposited in discrete target areas as a protein microarray, and thereafter subjecting said protein microarray to laser desorption/ionisation mass spectrometry.  
     
     
         127 . A method as claimed in  claim 126 , wherein said laser desorption/ionisation mass spectrometry is matrix assisted laser desorption/ionisation mass spectrometry (MALDI).  
     
     
         128 . A method as claimed in  claim 127 , wherein energy-absorbing molecules are deposited over the whole of said target surface or in registration with said discrete target areas in which protein has been captured.  
     
     
         129 . A method as claimed in  claim 128 , wherein said energy-absorbing molecules are deposited in registration with said discrete target areas in which protein has been captured.  
     
     
         130 . A method as claimed in claims  128  or  129  in which said energy-absorbing molecules are deposited in a manner which denatures and thus unbinds the protein from the protein capturing moieties, leaving the denatured protein in close proximity to said protein capture moieties on the surface.  
     
     
         131 . A method as claimed in any of claims  128  or  129 , wherein said energy-absorbing molecules are present as a homogenous layer in each discrete target area in registration with the captured protein.  
     
     
         132 . A method as claimed in  claim 131 , wherein said homogenous layer is substantially continuous such that individual crystals are not visible at a 100 fold magnification and there are no visible gaps between neighbouring crystals.  
     
     
         133 . A method as claimed in  claim 131 , wherein said homogenous layer is of a substantially uniform depth such that there is no apparent variation in crystal size at 100 fold magnification.  
     
     
         134 . A method as claimed in  claim 128 , wherein said energy-absorbing molecules are deposited onto the surface in a non-aqueous solvent, and said non-aqueous solvent is then evaporated off.  
     
     
         135 . A method as claimed in  claim 134 , wherein said non-aqueous solvent is an organic solvent.  
     
     
         136 . A method as claimed in  claim 135 , wherein said organic solvent is acetone or butanone.  
     
     
         137 . A method as claimed in  claim 134 , wherein said non-aqueous solvent includes a modifier which controls the rate of evaporation, such that evaporation of the non-aqueous solvent occurs after the energy-absorbing molecules are deposited.  
     
     
         138 . A method as claimed in  claim 137 , wherein said modifier is glycerol, polyetheleglycol or thioglycerol.  
     
     
         139 . A method as claimed in  claim 128 , wherein said energy-absorbing molecules are deposited in a mixture of from 80-99.9%, preferably 99%, non-aqueous solvent, preferably acetone, to 20-0.1%, preferably 1%, modifier, preferably glycerol (vol/vol).  
     
     
         140 . A method as claimed in  claim 128 , wherein said energy-absorbing molecules comprise crystals of α-cyano-4-hydroxy-cinnamic acid, sinapinic acid, gentisic acid, nifidine, succinic acid, 1,8,9,-anthracenitriol, 3-Indoleacrylic acid, 2-(hydroxyphenylazo) benzoe-acid, 4-nitroanilin and combinations thereof.  
     
     
         141 . A method of analysis by laser desorption/ionisation mass spectrometry, said method comprising the steps of: 
 a) providing a probe as claimed in  claim 85;     b) contacting said probe with one or more proteins; and    c) performing laser desorption/ionisation mass spectrometry on the proteins on the surface of the probe.    
     
     
         142 . A method as claimed in  claim 141  which comprises, between steps b) and c), an additional step of removing unbound molecules from the probe by washing.  
     
     
         143 . A method as claimed in  claim 142 , wherein said one or more proteins are contained in a mixture of proteins.  
     
     
         144 . The method of  claim 141 , which method comprises a method for identifying a protein on the surface of the probe, said method comprising the additional steps of: 
 d) determining the mass of the protein molecule;    e) performing a digestion upon a replicate sample of said protein on a further probe or probe surface; and    f) performing laser desorption/ionisation mass spectrometry on the peptides resulting from step e) to identify said proteins.    
     
     
         145 . The method of  claim 141 , which method comprises a method of analysing the function of a protein on the surface of the probe and a molecule interacting with said protein, said method comprising the additional steps, instead of step c), of: 
 c) bringing a protein on the probe surface into contact with one or more test molecules;    d) removing unbound test molecules from the probe surface; and    e) performing laser desorption/ionisation mass spectrometry on the protein and any bound molecule to determine the identity of the protein and/or test molecule.    
     
     
         146 . A method as claimed in  claim 145 , wherein said test molecule is a small molecule, protein or nucleic acid.  
     
     
         147 . A method as claimed in  claim 141 , which method comprises a method of analysing the function of a protein, said method comprising the additional steps of: 
 d) bringing a protein on the probe surface into contact with one or more test substrates;    and    e) performing laser desorption/ionisation mass spectrometry on the protein and test substrates to determine the presence and/or identity of products of catalysis of said test substrates by the protein.