Proteomics based on selecting and quantifying cysteine containing peptidesby covalent chromatography

This paper describes a procedure in which cysteine containing peptides from tryptic digests of complex protein mixtures were selected by covalent chro matography based on thiol-disulfide exchange, identified by mass spectromet ry, and quantified by differential isotope labeling. Following disruption o f disulfide bridges with 2,2 ' -dipyridyl disulfide, all proteins were dige sted with trypsin and acylated with succinic anhydride. Cysteine containing peptides were then selected from the acylated digest by disulfide intercha nge with sulfhydryl groups on a thiopropyl Sepharose gel. Captured cysteine containing peptides were released from the gel with 25 mM dithiothreitol ( pH 7.5) containing 1 mM (ethylenedinitrilo)tetraacetic acid disodium salt a nd alkylated with iodoacetic acid subsequent to fractionation by reversed-p hase liquid chromatography (RPLC). Fractions collected from the RPLC column were analyzed by matrix-assisted laser desorption ionization mass spectrom etry. Based on isotope ratios of peptides from experimental and control sam ples labeled with succinic and deuterated succinic anhydride, respectively, it was possible to determine the relative concentration of each peptide sp ecies between the two samples. Peptides obtained from proteins that were up -regulated in the experimental sample were easily identified by an increase of the relative amount of the deuterated peptide. The results of these stu dies indicate that by selecting cysteine containing peptides, the complexit y of protein digest could be reduced and database searches greatly simplifi ed. When coupled with the isotope labeling strategy for quantification it w as possible to determine proteins that were up-regulated in plasmid bearing Escherichia coli when expression of plasmid proteins was induced. Up-regul ation of several proteins of E. coli origin was also noted. (C) 2001 Publis hed by Elsevier Science B.V.