Patent ID: 7354996

Claim:
A method for global quantitative analysis of protein comprising the steps of: (i) preparing two states of protein samples, a Protein sample I for analysis and a control Protein sample II; (ii) solubilizing the Protein sample I in a solution containing urea as a denaturing agent or in a solution containing guanidine hydrochloride as a denaturing agent, to obtain a solubilized Protein sample I, and separately solubilizing the Protein sample II in a solution containing urea as a denaturing agent or in a solution containing guanidine hydrochloride as a denaturing agent, to obtain a solubilized Protein sample II; (iii) subjecting the solubilized Protein sample I to modification reaction using either one of 2-nitro[ 13 C 6 ]benzenesulfenyl chloride and 2-nitro[ 12 C 6 ]benzenesulfenyl chloride, to obtain a modified Protein sample I, and separately subjecting the solubilized Protein sample II to modification reaction using the other one of 2-nitro[ 13 C 6 ]benzenesulfenyl chloride and 2-nitro[ 12 C 6 ]benzenesulfenyl chloride, to obtain a modified Protein sample II; (iv) mixing and desalting the modified Protein sample I and the modified Protein sample II, to obtain a desalted protein sample mixture; (v) resolubilizing the desalted protein sample mixture by using urea or guanidine hydrochloride, to obtain aresolubilized protein sample mixture; (vi) reducing and alkylating the resolubilized protein sample mixture, to obtain a reduced and alkylated protein sample mixture; (vii) subjecting the reduced and alkylated protein sample mixture to trypsin digestion in the presence of urea or guanidine hydrochloride, to obtain a peptide mixture containing modified peptide fragments and unmodified peptide fragments; (viii) separating the peptide mixture using a media having a phenyl group, to obtain enriched modified peptide fragments; and (ix) subjecting the enriched modified peptide fragments to mass spectrometry.