| Quantitative proteomics is set to play a pivotal role in the discovery of diagnostic or prognostic protein markers, for the detection of new therapeutic targets, and as a powerful tool to further our understanding of basic biological processes and mechanisms. The realization of these expectations will rely on the development of highly sensitive, efficient and reliable methods for quantitative proteome analysis. This thesis work is devoted to the development and applications of novel strategies for quantitative proteome analysis based on global stable isotope labeling and microbore liquid chromatography (LC) matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight (QqTOF) mass spectrometry (MS).; Compared to conventional LC electrospray ionization (ESI) based quantitative proteomic strategies, LC-MALDI based strategies allow global quantification and selective identification of differentially expressed proteins between two proteome samples, such as two cell lines. It was demonstrated that differential isotope dimethyl labeling of peptides and two-dimensional LC-MALDI QqTOF MS allowed accurate quantification and identification of differentially expressed proteins. Setting a relative abundance ratio of greater than 2-fold was demonstrated to be a very stringent threshold to quantify and identify differentially expressed proteins between two cultured cell lines. LC-MALDI also allowed the use of microbore instead of microcapillary LC for peptide separation, providing higher sample loading, accurate protein quantification due to the increased signal-to-noise ratio of peptide pairs, and the quantification of protein mixtures with a concentration dynamic range of as high as 1 x 104.; It was demonstrated that a novel isotope labeling technique, 2MEGA, involving N-terminal dimethylation (2ME) after lysine guanidination (GA), offered several desirable features, including: simple experimental procedure, the use of inexpensive and commercially available reagents, and negligible isotope effect on reversed-phase separations. In addition to its applicability to quantitative analysis, LC-MALDI MS combined with 2MEGA was successfully used to identify proteins that included polymorphic variants, phosphopeptides and low abundance proteins in complex samples, such as the whey fraction of bovine milk. It was also demonstrated that the enhanced a1 or a1-related peaks in both MALDI and ESI tandem mass spectra of 2MEGA labeled peptides provided additional information that reduced false positive identifications. |
