Development and Applications of Quantitative Mass Spectrometric Methods for Proteome and Protein Analysis

Quantitative proteomics has been increasingly recognized as a key research topic in proteomics research, as it enumerates changes of proteins' expression level in a bio-system. Quantitative proteomics provides an important way to discover biomarkers for disease diagnosis and prognosis, and also to understand biological processes and mechanisms. This thesis describes the method optimization in shotgun proteomic sample preparation and 2-MEGA (dimethylation after guanidation) labeling, which is known as a stable isotopic labeling technique. In this work, a LC-UV quantification method was developed to evaluate sample integrity in shotgun proteome sample preparation. Based on the quantification results obtained with this method, MS analysis parameters could be optimized to ensure better proteome coverage in profiling a proteomic sample. Also, in shotgun proteomic sample preparation for quantification, the compatibility of 2-MEGA labeling chemistry with commonly used cell lysis buffer, salts and detergents was evaluated to ensure better than 95% correct labeling of the peptides. In all tested reaction conditions, the most common side reaction products were N-terminal guanidination (~2%) for glycine and alanine N-terminal peptides. Various protein sample preparation methods were found to be compatible with 2-MEGA labeling.;In addition to comparative proteomic quantification, my thesis work also included the development of a novel MS-based absolute quantification method for a mixture of N-truncated protein and intact protein. It was based on dansyl labeling reaction on N-terminal free amine of proteins. Microwave-assisted acid hydrolysis (MAAH) were performed after dansylation to release N-terminal amino acids with the dansyl label, which were quantified on MS with internal standards of dansyl labeled amino acids.;Taken together, the thesis work developed several new analytical methods to facilitate protein quantification and illustrated an increasingly important role of quantitative mass spectrometry in meeting the bioanalytical challenges in protein and proteome characterization.;After the optimization of the sample preparation process, the 2-MEGA labeling method was applied to comparative analysis of HER2 normal and overexpressing MCF-7 human breast cancer cell lines, as well as plasma samples of pig model collected at different stages and treatments of deep tissue injury (DTI). In the breast cancer cell experiment, 4 potential biomarkers with different expression level change in HER2 normal and HER2 overexpressing cell lines were identified. In the pig plasma sample analysis, a number of differentially expressed proteins were identified that may potentially be used as biomarkers for diagnosing and studying DTI. In the future, more biological evaluation work needs to be done to reveal more information for their prognostic values.