Development and Applications of Mass Spectrometric Methods for Proteome Analysis and Protein Sequence Characterization

Abstract

Mass spectrometry (MS) is widely used in proteomic work for protein identification and characterization. A key to the success of MS for protein analysis is related to sample preparation. Current sample preparation methods do not meet all the needs of protein or proteome analysis. My thesis work is devoted to the development and application of new or improved protein sample preparation methods for MS analysis. These methods are focused on three aspects: microbore LC-UV for quantification of peptides generated from the proteins extracted from a small number of cells, 2-MEGA (N-terminal dimethylation after lysine guanidination) isotope labeling for proteome quantification, and microwave-assisted acid hydrolysis (MAAH) for protein sequence analysis. It is shown that microbore LC-UV is a robust technique for quantifying peptides down to about 40 ng, as well as for removing salts for LC-MS/MS analysis of peptides and this method can be used to gauge the sample integrity during the proteomic sample preparation in dealing with small number of cells. An automated 2-MEGA labeling method utilizing a commercial liquid handler was developed to minimize variability from sample handling during the labeling reaction for high throughput applications. To find biomarkers of breast cancer, the 2-MEGA labeling method was applied to the breast cancer tissues for relative proteomic comparison with the normal breast tissues. 119 proteins were differentially expressed in all three tumor samples and some of these proteins can potentially be verified and validated as biomarkers. To provide a detailed characterization of a protein of interest including amino acid substitution and modifications, MAAH MS was further developed to improve its performance and applicability. Hydrolysis performed in an optimized procedure using a commercial microwave oven was found to be comparable with that in a household microwave oven. A method of characterizing terminal peptides of a protein based on HCl MAAH of proteins, LC fractionation of the hydrolysates and LC-ESI MS/MS analysis of the low molecular weight peptides, was developed. In addition, proteins separated by gel can be hydrolyzed by MAAH to analyze their protein sequences. Both electroelution of proteins from a gel and in-gel MAAH were studied for analyzing gel-separated proteins.