Development And Application Of Dianhydride Bifunction Reagent Chemical Modification-assisted Proteomic Strategies

The mass spectrometry based methods with a stable isotope as the internal standard in quantitative proteomics have been developed so quickly in recent years. There is a great need of improvement of proteomics strategies and construction of new techniques and methods due to such disadvantages of stable isotope reagents as high price and synthetic difficulties. In this research, we developed a series of new proteomics methods based on dianhydride bifunction reagent assisted modification, and evaluated the methods by their application in the real biosamples.In chapter I, a new method is developed based on metal chelates and labeling of primary amines in peptides for relative quantification and identification of proteome. To introduce the rare earth metal chelate tag, the bicyclic anhydride diethylenetriamine-N, N, N', N", N"-pentaacetic acid (DTPA) is covalently coupled to the peptide amine, and then chelated with the rare metal. After the peptides are labeled with the rare metal ions of Y and Tb, the tagged peptides are mixed and analyzed by LC-MS/MS. Compared with current proteomics quantititave methods, the strategy has the advantegies of universally labeling to all primary amines, relatively cheaper and easy to get with the DTPA dianhydride, and the improved confidence level of protein identification by MS with cleaner fragmentation (only y-series ions). The related results has been published in Analytical Chemistry (2006, 78,6614-6621).In chapter II, we develope a non-gel-based, dual ¹⁸O labeling strategy to improve the quantitation of target proteins in proteomic analyses, which combines chemical ¹⁸O labeling and chemical and enzyme-catalyzed ¹⁸O labeling by using DTPA. In the first ¹⁸O labeling method (chemical ¹⁸O labeling) of the dual strategy, one functional group of DTPA is covalently coupled to the primary amines of peptides, and ¹⁸O is incorporated at the other functional group by hydrolysis. In the second ¹⁸O labeling method (chemical and enzyme-catalyzed ¹⁸O labeling), chemical ¹⁸O labeling and enzyme-catalyzed ¹⁸O labeling of the carboxyl termini of peptides are combined. In contrast to current proteolytic ¹⁸O labeling methods, there is no ¹⁸O-to-¹⁶O back exchange in the first method and no isotope peaks in MS in the second method. The combination of chemical and proteolytic ¹⁸O labeling improves the confidence of the quantitation results and simplifis the sample preparation.In chapter III, we applies dual ¹⁸O labeling quantitative proteomics strategy to biological samples of Saccharomyces cerevisiae to investigate the feasibility, accuracy and reliability of the method, and to further understand different biological process between YHB1 deleted strain and wild type one under normal culture circumstance.In chapter IV, by using dual 0 labeling quantitative proteomics strategy, we try to find out the differential expression of proteins under the stimulation of NO donor between YHB1 deleted strain and the wild type one. A series of proteins are found up- or down-regulated by the stimulation of NO, which may account for NO-related signaling pathway and regulation mechanism in the absence of YHB1.In chapter V, we develope a new method for the enrichment of N-peptide labeled by DTPA to address such problems as high complexities of peptide mixtures from samples, low confident level of protein identification, and difficulties in obtaining terminal information of proteins. The preliminary result showed that the method is promising to be utilized in the N-termini determination of the proteins in large scale.