Comparative proteomics based on global internal standard labeling technology
Abstract
The concept of studying comparative proteomics through stable isotope labeling was evaluated in this research. The work described here started with the development of global internal standard technology (GIST) for quantification of relative changes in protein expression. The GIST protocol involved tryptic digestion of proteins from control and experimental samples followed by differential isotopic labeling of the resulting peptides with N-acetoxysuccinimide and N-acetoxy-2H3-succinimide separately, mixing the labeled control and experimental samples, fractionation by reversed phase chromatography, and isotope ratio analysis by mass spectrometry. The second portion of the work focused on the development of a new metal affinity chromatographic method to be used with GIST for simplification of complex mixtures through targeting histidine-containing peptides as part of signature peptide approach to comparative proteomics. A further study was directed toward the development of a high throughput method for proteomics using multiplexed global internal standard technology (mGIST). By applying mGIST, quantitative comparisons between samples from three different cell states were achieved simultaneously. N-acetoxysuccinimide, N-acetoxy-1, 2-13C2-succinimide and N-acetoxy-1, 2-13C2-2H3-succinimide were used to differentially derivatized peptides from three cell states. Finally, the efficacy of GIST for relative peptide quantification in a tandem mass spectrometer was examined. The results indicate that peptides differentially labeled with N-hydroxysuccinimide activated acetate and trideuteroacetate, propionate and 13C3- or trideuteropropionate, propionate and pentadeuteropropionate can be used to quantify and facilitate the identification of tryptic peptides as well as to aid the sequencing of peptide fragment ions generated by collisionally activated dissociation.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry|Biochemistry|Pharmacology
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