Global internal standard technology for comparative proteomics.

Abstract

The work described in this paper tests the efficacy of a global isotope labeling (global internal standard technology, GIST) strategy for quantification in proteomics. Using GIST, overexpression of beta-galactosidase in Escherichia coli was identified and quantified. The GIST protocol involved tryptic digestion of proteins from control and experimental samples followed by differential isotopic labeling of the resulting tryptic peptides, mixing the differentially labeled control and experimental digests, fractionation of the peptide mixture by reversed-phase chromatography, and isotope ratio analysis by mass spectrometry. N-Acetoxysuccinimide and N-acetoxy-[2H3]succinimide were used to differentially derivatize primary amino groups in peptides from experimental and control samples, respectively. The relative concentration of isotopically labeled peptides was determined by isotope ratio analysis with both matrix-assisted laser desorption ionization mass spectrometry and tandem mass spectrometry (MS-MS). Peptide masses and sequences obtained by MS-MS were used to identify proteins. MS-MS was found to be uniquely suited for isobaric peptide quantification.