Advances in the development of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) instrumentation have made this technique a versatile analytical tool to identify, characterize and quantify biomolecules including peptides, proteins, lipids, nucleic acids, oligosaccharides and other metabolites. However, based on the individual physicochemical properties of various biomolecules, biomolecular MS or MS/MS on its own may not necessarily give the desired analytical information. Therefore, chemical labeling strategies which alter the behavior of analytes with respect to their ionization, fragmentation and mass analysis are commonly used to facilitate MS-based analysis of biomolecules. This dissertation focuses on the development of biomolecular chemical labeling strategies for lipids, peptides and proteins, to provide improved capabilities for MS-based qualitative and quantitative analysis. Structural labeling via gas phase ion chemistry provides a convenient and rapid modification method for structural and reactivity characterization of modified biomolecular ions. Here, a novel photo-induced inter-molecular gas-phase cross-linking reaction has been developed to investigate the cross-linking reactivity of individual triacylglyceride (TG) molecules as a function of their structures. Ultraviolet photodissociation tandem mass spectrometry (UVPD-MS/MS) of non-covalent complex ions consisting of TG dimers and protonated diiodoaniline resulted in the formation of multiple cross-linked TG products via homolysis of carbon-iodine bonds, hydrogen abstraction and radical recombination. The efficiency of the UVPD reaction depended on the number of unsaturation sites present within the TG lipids. For MS-based quantification, an approach for the multiplexed relative quantification of aminophospholipids from within two different crude lipid extracts was developed. Relative quantification at the ‘sum composition’ and/or ‘molecular lipid’ levels was achieved using high resolution/accurate mass MS/MS by ratiometric measurement of pairs of ‘reporter’ ions formed via the neutral loss from isobaric stable isotope-labeled d6-‘heavy’ and d6-‘light’ S,S′-dimethylthiobutanoylhydroxysuccinimide and iodine/methanol derivatized aminophospholipid ions. In addition, absolute quantification of full length parathyroid hormone (PTH 1-84), a clinical protein biomarker of secondary hyperparathyroidism, and its in vivo oxidized and truncated variants was achieved using a dual stable isotope-labeled internal standard approach coupled with immunocapture and high resolution LC-MS and MS/MS. Analysis of clinical PTH samples using this strategy revealed that no oxidation or PTH 7-84 occurred in vivo. However, several novel sites of in vivo PTH truncation were discovered. At last, stable isotope-containing dimethyl labeling and multi-dimensional LC-MS/MS were applied for proteomic profiling of human RPMI-8226 cells treated with competitive (i.e., Bortezomib) and non-competitive (i.e., TCH-013) proteasome inhibitors to evaluate their distinct mechanisms of action. Four proteins closely related to the regulation of mitochondrial functions and growth and division of cancer cells were observed to be selectively down-regulated after TCH-013 treatment compared to Bortezomib or vehicle control treatment.
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