Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation

We have demonstrated the use of per-methyl esterification of peptides for r elative quantification of proteins between two mixtures of proteins and aut omated de novo sequence derivation on the same dataset, Protein mixtures fo r comparison were digested to peptides and resultant peptides methylated us ing either d0- or d3-methanol, Methyl esterification of peptides converted carboxylic acids, such as are present on the side chains of aspartic and gl utamic acid as well as the carboxyl terminus, to their corresponding methyl esters, The separate d0- and d3-methylated peptide mixtures were combined and the mixture subjected to microcapillary high performance liquid chromat ography/tandem mass spectrometry (HPLC/MS/MS). Parent proteins of methylate d peptides were identified by correlative database searching of peptide tan dem mass spectra, Ratios of proteins in the two original mixtures could be calculated by normalization of the area under the curve for identical charg e states of d0- to d3-methylated peptides, An algorithm was developed that derived, without intervention, peptide sequence de novo by comparison of ta ndem mass spectra of d0- and d3-peptide methyl esters, Copyright (C) 2001 J ohn Wiley & Sons, Ltd.