Based On Mass Spectrometry Of Histone Modification After Translation And Quantitative Proteomics Research

The histone post translational modifications (PTMs) is one of the research hot spots of epigenetics, and some MS-based researches regarding identification and discovery of histone post translational modification sites are attracting more and more attention of scientists. Meanwhile, quantitative proteomics, as an important branch of proteomics, some new method researches of which play a critical role in the development of proteomics. Based on the mass spectrometry, this research work established some identification methods of histone post translational modifications, which can be applied in the discovery of new post translational modification sites. Besides, a novel in vivo terminal amino acid labeling (IVTAL) method was also established, and an analysis and optimization software (ITMSQ) for experimental data has been designed, edited, verified and applied. In order to popularize IVTAL, some works were conducted to get an optimized and global in vivo terminal amino acid labeling method (G-IVTAL).Main contents in the thesis are as follows:1) The basic definition of histone, histone variant, type and function of histone PTMs, challenges and main methods of histone PTMs analysis, especially the MS-based analytical methods have been summarized or introduced. Additionally, the development status of quantitative proteomics, the principles, advantages and disadvantages, and application of various quantitative proteomics methods, some newly developed quantitative proteomics methods, and the related data analysis software were introduced.2) The concentrated sulphuric acid extraction method was used to successfully realize the histone extraction from cells. The on-line high resolution RPLC-LTQ-Orbitrap histone molecular weight identification method was used to observe the distribution of histone variants and their PTMs on the level of proteins, and was also applied in the comparison of multiple liver cancer cells. The multiple enzymes and RPLC-high resolution LTQ-Orbitrap combinational method has been established to identify the PTM sites of histones at the premise of obtaining the overall histone PTMs distribution. This method was also applied in the human liver cancer cell QGY-7703and a new methylated site R67in H4was found. Meanwhile, the pressure bomb-HILIC-LTQ-Orbitrap combinational method has also been used to analyze for the H3PTMs of the human liver cancer cell QGY-7703and MALDI-based specific marker ions in the low mass range have been used to explore the sites of monomethylation, dimethylation, trimethylation and acetylation.3) Based on the present quantitative proteomic methods, in order to resolve the difficulty of increased complexity and decreased sensitivity of MS1-based mass difference quantitative methods, and problems of repression effect existed in the isobaric mass in MS1, a novel method of in vivo terminal amino acid labeling has been established. The core of this strategy is a set of heavy amino acid13C6-arginine and13C6-lysine and specific endoproteinase Lys-N and Arg-C that yield some labeled isobaric peptides by cell culture and enzymatic digestion, which are indistinguishable in MS scan but exhibit multiple MS/MS reporter b, y ion pairs in a full mass range that support quantitation. Relative quantification of cell states can be achieved by calculating the intensity ratios of the corresponding reporter b, y ions in MS/MS scan. Compared to some traditional methods, in which a peptide is quantified only by a quantitative ratio, IVTAL adapts multiple b, y fragment ion pairs to quantify a peptide. Therefore, the quantitation information of peptide will be more accurate and reliable. IVTAL, as a highly accurate and reliable quantitative proteomic approach, is expected to be compatible with any cell culture system and to be especially effective for the analysis of multiple post translational modification sites in one peptide. In addition, this method can realize the identification and quantitation in MS2, and b, y fragment ion pairs can also provide some accurate complimentary information for qualitative analysis.4) Based on IVTAL, search engine of SEQUEST software, Perl software and Matlab software, a new software, termed isobaric tandem MS quantitative (ITMSQ), was developed for analysis and optimization of isobaric tandem MS quantitative data. This software mainly achieves quantification information of peptides and proteins by calculating the intensity ratios of the corresponding reporter b, y ion pairs in MS/MS scan, and ITMSQ can be applied in all the isobaric MS2quantitative methods including iMSTIQ and IPTL. In addition, aimed at the MS2spectrum of b, y fragment ion pairs co-fragmentation, an optimized module is added to the software, greatly increasing the data scale of identification and quantitation and ensuring the accuracy and reliability of quantitative data. The effectiveness of the proposed software was successfully tested by the MS data from IVTAL-labeled HeLa samples, and the up and down regulated boundaries of IVTAL was obtained by statistically analyzing quantitative data. The effects of mass error and ion intensity of b, y ion pairs on the accuracy of IVTAL in the condition of SEQUEST search and b, y ion pairs extraction were investigated, respectively.5) In order to make the IVTAL method to be applied more simply, conveniently and widely in the field of proteomics research, IVTAL was further optimized and combined trypsin digestion and N terminal dimethylation labeling to form a global in vivo terminal amino acid labeling(G-IVTAL).Two cell lines were cultured in the medium of normal arginine and lysine or heavy15N4-arginine and13C615N2-lysine, and the cell lysates were digested by trypsin and labeled by deuterium formaldehyde or normal formaldehyde to produce isobaric peptides in MS1and b, y fragment ion pairs in MS2, which could be used for the protein quantitation. Compared to IVTAL, G-IVTAL can provide more qualitative and quantitative information due to a full use of digested peptides generated by trypsin.