Proteomics based on selecting and quantifying cysteine containing peptides by covalent chromatography.
This paper describes a procedure in which cysteine containing peptides from tryptic digests of complex protein mixtures were selected by covalent chromatography based on thiol-disulfide exchange. identified by mass spectrometry, and quantified by differential isotope labeling. Following disruption of disulfide bridges with 2,2'-dipyridyl disulfide, all proteins were digested with trypsin and acylated with succinic anhydride. Cysteine containing peptides were then selected from the acylated digest by disulfide interchange 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 and alkylated with iodoacetic acid subsequent to fractionation by reversed-phase liquid chromatography (RPLC). Fractions collected from the RPLC column were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Based on isotope ratios of peptides from experimental and control samples labeled with succinic and deuterated succinic anhydride, respectively, it was possible to determine the relative concentration of each peptide species 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 studies indicate that by selecting cysteine containing peptides, the complexity of protein digest could be reduced and database searches greatly simplified. When coupled with the isotope labeling strategy for quantification it was possible to determine proteins that were up-regulated in plasmid bearing Escherichia coli when expression of plasmid proteins was induced. Up-regulation of several proteins of E. coli origin was also noted.
Wang
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S
Department of Chemistry, Purdue University, Lafayette, IN 47907, USA.
Regnier
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FE
D000478:Alkylation; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D003545:Cysteine / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D020543:Proteome; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010455:Peptides; D020543:Proteome; D011994:Recombinant Proteins; D003545:Cysteine