Selective analysis of protein N-terminal acetylation and its application to study the acetylation profiling of HepG2 cells upon oxidative stress

• Main Authors: Sin-Hong Chen, 陳信宏
• Other Authors: Jue-Liang Hsu
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/89801419871687606600

碩士 === 國立屏東科技大學 === 生物科技研究所 === 100 === N-terminal acetylation is one of the most common and abundant protein modifications in eukaryotes and it is estimated that about 85% of mammalian proteins and 60% of yeast proteins are acetylated at protein N-termini co-translationally or post-translationally. Several studies have linked it to normal cell function and cancer development. However, still little is known about the function and regulation of the human N-terminal acetylation system, which may be mainly due to the lack of a robust platform to efficiently analyze and profile the protein N-terminal acetylation in a large scale. In this study, strong cation exchange chromatographic method, stable-isotope labeling, high performance LC-MS/MS (liquid chromatography-tandem mass spectrometry) and database matching were integrated for a large-scale study of protein N-terminal acetylation. Using this approach, several hundreds of N-terminal acetylation sites were identified, including the co-translationally modified residues and post-translationally modified residues which were underwent enzymatic cleavage. In this study, we mainly focused on the improvement to increase the numbers of identified N-acetylated peptides. By comparison with SCX-only approach, our method has much better specificity for enrichment of N-acetylated peptides. Moreover, the degree of protein N-terminal acetylation can be simultaneously obtained when the deuterium labeled acetic N-hydroxyacetamide was incorporated in this approach. This method was further introduced to the study of comprehensive N-terminal acetylation profiling of HepG2 cells under oxidative stressed by tert-butyl peroxide. The degrees of protein N-terminal acetylation of some proteins were varied which indicated that the N-terminal acetylation of these proteins may be involved in the oxidative stress.