Affinity selected signature peptides for protein identification and quantification

Abstract

A method for protein identification in complex mixtures that utilizes affinity selection of constituent proteolytic peptide fragments unique to a protein analyte. These “signature peptides” function as analytical surrogates. Mass spectrometric analysis of the proteolyzed mixture permits identification of a protein in a complex sample without purifying the protein or obtaining its composite peptide signature.

1 .- 62 . (canceled) 
     
     
         63 . A method for identifying a protein in a sample comprising a plurality of proteins, the method comprising:
 providing peptides derived from fragmentation of proteins in a sample comprising a plurality of proteins, wherein at least one peptide derived from the protein to be identified comprises at least one affinity ligand;   contacting the peptides with a capture moiety to select peptides comprising the affinity ligand;   fractionating the selected peptides to yield a plurality of peptide fractions;   subjecting the peptides in at least one peptide fraction to mass spectrometric analysis to detect at least one peptide derived from the protein to be identified; and   identifying the protein from which the detected peptide was derived.   
     
     
         64 . The method of  claim 63  wherein the detected peptide is a signature peptide of the protein to be identified, the method further comprising determining the mass of the signature peptide and using the mass of the signature peptide to identify the protein from which the detected peptide was derived. 
     
     
         65 . The method of  claim 63  further comprising determining the amino acid sequence of the detected peptide and using the amino acid sequence of the detected peptide to identify the protein from which the detected peptide was derived. 
     
     
         66 . The method of  claim 63  further comprising, prior to contacting the peptides with the capture moiety, covalently attaching at least one affinity ligand to at least one peptide derived from the fragmentation of the proteins. 
     
     
         67 . The method of  claim 63  further comprising, prior to fragmenting the proteins, covalently attaching at least one affinity ligand to at least one protein in the sample. 
     
     
         68 . The method of  claim 63  further comprising reducing and alkylating the proteins with an alkylating agent prior to fragmenting the proteins. 
     
     
         69 . The method of  claim 68  wherein the at least one affinity ligand is covalently attached to the alkylating agent. 
     
     
         70 . The method of  claim 63  wherein the at least one affinity ligand is covalently attached to an amino acid of the peptide selected from the group consisting of cysteine, tyrosine, tryptophan, histidine and methionine. 
     
     
         71 . The method of  claim 63  wherein the affinity ligand comprises a moiety selected from the group consisting of a peptide antigen, a polyhistidine, a biotin, a dinitrophenol, an oligonucleotide and a peptide nucleic acid. 
     
     
         72 . The method of  claim 63  wherein at least one peptide comprises an endogenous affinity ligand. 
     
     
         73 . The method of  claim 72  wherein the endogenous affinity ligand comprises a phosphate group or a carbohydrate. 
     
     
         74 . The method of  claim 73  wherein the endogenous affinity ligand comprises a phosphate group, and wherein contacting the peptides with a capture moiety comprises contacting the peptides at acidic pH with a cationic support surface. 
     
     
         75 . The method of  claim 72  wherein the endogenous affinity ligand comprises a cysteine or a histidine. 
     
     
         76 . The method of  claim 72  wherein the endogenous affinity ligand comprises an antigenic amino acid sequence. 
     
     
         77 . The method of  claim 63  further comprising attaching a plurality of affinity ligands, each to at least one protein or peptide, and contacting the peptides with a plurality of capture moieties to select peptides comprising at least one affinity ligand. 
     
     
         78 . The method of  claim 63  further comprising fragmenting the proteins in the sample to yield the peptides. 
     
     
         79 . The method of  claim 78  wherein the proteins are fragmented using an enzyme selected from the group consisting of trypsin, chymotrypsin, gluc-C, endo lys-C, pepsin, papain, proteinase K, carboxypeptidase, calpain and subtilisin. 
     
     
         80 . The method of  claim 63  wherein fractionating the selected peptides comprises subjecting the selected peptides to at least one separation technique selected from the group consisting of reversed phase chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, capillary gel electrophoresis, capillary zone electrophoresis and capillary electrochromatography, capillary isoelectric focusing, immobilized metal affinity chromatography and affinity electrophoresis. 
     
     
         81 . The method of  claim 63  wherein the sample comprises at least about 100 proteins. 
     
     
         82 . The method of  claim 63  wherein using the mass of the signature peptide to identify the protein from which the signature peptide was derived comprises comparing the mass of the signature peptide with the masses of reference peptides derived from putative proteolytic cleavage of a plurality of reference proteins in a database, wherein at least one reference peptide comprises at least one affinity ligand. 
     
     
         83 . The method of  claim 82  wherein peptides derived from fragmentation of the plurality of reference proteins are, prior to comparing the mass of the signature peptide with the masses of the reference peptides, computationally selected to exclude reference peptides that do not contain an amino acid upon which the affinity selection is based. 
     
     
         84 .- 99 . (canceled)