METHODS FOR PEPTIDE ANALYSIS USING MASS SPECTROMETRY

摘要

The invention pertains to ("CAMPA") methods for the preparation of a polypeptide sample for mass spectrometric analysis from a polypeptide, said method comprising the steps of: (a) treating the polypeptide with an oxidation reagent, to convert disulfide groups and/or sulfhydryl groups to sulfonic acid groups; (b) subjecting the polypeptide, optionally the polypeptide produced in step (a), to site-specific proteolysis, to yield proteolytic peptide fragments which have: (A) a C-terminus having a basic amino acid which is located at the C-terminus, or proximal to the C-terminus; and (B) an N-terminus having a sulfonic acid amino acid which is located at the N-terminus, or proximal to the N-terminus; wherein said basic amino acid is located on the C-terminal side of said sulfonic acid amino acid; and wherein said sulfonic acid amino acid is located on the N-terminal side of said basic amino acid. The invention also pertains to ("CAPPA") methods, in which step (a) is replaced with the following step: (a*) in order: (a*-1) optionally deglycosylating the polypeptide, to remove O-glycan groups; (a*-2) reducing cystine residues, to form cysteine residues; (a*-3) alkylating cysteine residues, to form protected cysteine residues; (a*-4) removing phosphate groups by -elimination, to form phosphate-eliminated amino acid residues; and (a*-5) adding sulfonic acid groups by sulfite addition at the phosphate-eliminated amino acid residues, to form sulfonic acid amino acid residues. The invention also pertains to ("CAGPA") methods, in which step (a) is replaced with the following step: (a**) in order: (a**-1) optionally dephosphorylating the polypeptide, to remove phosphate groups; (a**-2) reducing cystine residues, to form cysteine residues; (a**-3) alkylating cysteine residues, to form protected cysteine residues; (a**-4) removing O-glycan groups by -elimination, to form O-glycan-eliminated amino acid residues; (a**-5) adding sulfonic acid groups by sulfite addition at the O-glycan-eliminated amino acid residues, to form sulfonic acid amino acid residues. The invention also pertains to methods of performing mass spectrometric analysis and methods of identifying a polypeptide which employ the above methods, and apparatus configured to perform such methods.