Methods for high throughput and quantitative proteome analysis

Abstract

The invention provides methods for identifying and quantifying polypeptides in a sample. The methods include the steps of labeling peptides in a polypeptide sample with an isotope tag; adding a plurality of peptide standards to the polypeptide sample, wherein the peptide standards are labeled with an isotopically distinct version of the isotope tag; resolving the labeled sample and standard peptides into a plurality of fractions; analyzing the resolved fractions using mass spectrometry; identifying an isotope-tagged sample peptide in an analyzed fraction; and determining the amount of the identified isotope-tagged sample peptide in the analyzed fraction by comparison to the amount of isotope tagged standard peptide in the same fraction.

1 . A method for identifying and quantifying polypeptides in a sample, comprising the steps of:
 (a) labeling peptides in a polypeptide sample with an isotope tag;   (b) adding a plurality of peptide standards to said polypeptide sample, wherein said peptide standards are labeled with an isotopically distinct version of said isotope tag;   (c) resolving said labeled sample and standard peptides into a plurality of fractions;   (d) analyzing said resolved fractions using mass spectrometry;   (e) identifying an isotope-tagged sample peptide in an analyzed fraction; and   (f) determining the amount of the identified isotope-tagged sample peptide in said analyzed fraction by comparison to the amount of isotope tagged standard peptide in the same fraction.   
     
     
         2 . The method of  claim 1 , wherein said plurality of fractions is deposited onto a mass spectrometry sample plate. 
     
     
         3 . The method of  claim 1 , wherein a known absolute amount of each of said peptide standards is added to said polypeptide sample. 
     
     
         4 . The method of  claim 1 , wherein said polypeptide sample is cleaved with a protease. 
     
     
         5 . The method of  claim 4 , wherein said protease is trypsin. 
     
     
         6 . The method of  claim 1 , wherein said sample is derived from a body fluid selected from the group consisting of blood, plasma, cerebrospinal fluid, urine, saliva, seminal plasma, and pancreatic juice. 
     
     
         7 . The method of  claim 6 , wherein said sample is derived from serum. 
     
     
         8 . A method for quantifying polypeptides in a sample, comprising the steps of:
 (a) labeling peptides in a polypeptide sample with an isotope tag;   (b) adding a known absolute amount of a plurality of peptide standards to said polypeptide sample, wherein said peptide standards are labeled with an isotopically distinct version of said isotope tag;   (c) resolving said labeled sample and standard peptides into a plurality of fractions;   (d) analyzing said resolved fractions using mass spectrometry;   (e) identifying an isotope-tagged sample peptide in an analyzed fraction; and   (f) determining the amount of the identified isotope-tagged sample peptide in said analyzed fraction by comparison to the amount of isotope tagged standard peptide in the same fraction.   
     
     
         9 . The method of  claim 8 , wherein said plurality of fractions is deposited onto a mass spectrometry sample plate. 
     
     
         10 . The method of  claim 8 , wherein said polypeptide sample is cleaved with a protease. 
     
     
         11 . The method of  claim 10 , wherein said protease is trypsin. 
     
     
         12 . The method of  claim 8 , wherein said sample is derived from a body fluid selected from the group consisting of blood, plasma, cerebrospinal fluid, urine, saliva, seminal plasma, and pancreatic juice. 
     
     
         13 . The method of  claim 12 , wherein said sample is derived from serum. 
     
     
         14 . A method for identifying and quantifying splice isoforms of polypeptides in a sample, comprising the steps of:
 (a) labeling peptides in a polypeptide sample with an isotope tag;   (b) adding a plurality of peptide standards to said polypeptide sample, wherein said peptide standards are labeled with an isotopically distinct version of said isotope tag and wherein said plurality of peptide standards comprises at least one peptide corresponding to a common amino acid sequence of a splice isoform of a polypeptide and at least one peptide corresponding to an amino acid sequence that differs between two splice isoforms of said polypeptide;   (c) resolving said labeled sample and standard peptides into a plurality of fractions;   (d) analyzing said resolved fractions using mass spectrometry;   (e) identifying an isotope-tagged sample peptide in an analyzed fraction; and   (f) determining the amount of the identified isotope-tagged sample peptide in said analyzed fraction by comparison to the amount of isotope tagged standard peptide in the same fraction.   
     
     
         15 . The method of  claim 14 , wherein said plurality of fractions is deposited onto a mass spectrometry sample plate. 
     
     
         16 . The method of  claim 14 , wherein a known absolute amount of each of said peptide standards is added to said polypeptide sample. 
     
     
         17 . The method of  claim 14 , wherein said polypeptide sample is cleaved with a protease. 
     
     
         18 . The method of  claim 17 , wherein said protease is trypsin. 
     
     
         19 . The method of  claim 14 , wherein said sample is derived from a body fluid selected from the group consisting of blood, plasma, cerebrospinal fluid, urine, saliva, seminal plasma, and pancreatic juice. 
     
     
         20 . The method of  claim 19 , wherein said sample is derived from serum.