Acetylated Protein Identification Study

Lysine acetylation is a reversible and highly regulated posttranslational modification. It mainly occurs at the site of S-NH2 of lysine in the protein. The modification regulates diverse protein properties including DNA-protein interactions, subcellular localization, transcriptional activity, and protein stability. In addition to its important roles in fundamental biology, lysine acetylation and its regulatory enzymes (histone acetyltransferases and histone deacetylases ) are intimately linked to aging and several major diseases such as cancer, neurodegenerative disorders, and cardiovascular diseases. So the identification of lysine acetylation is of great biological significance. Most of the known acetylated proteins have been identified by radioactive detection or immunedetection, however, both radioactive detection and immunodetection provide evidence for acetylation but do not give information about the number of acetylation sites in a protein. In contrast, mass spectrometric measurement containing ESI-MS and MALDI-MS not only allows posttranslational modification characterization but also reveals the number of acetyl groups attached to the protein.This study aims at the application of ESI-MS and MALDI-MS in the identification of acetylation of mouse liver. The first chapter gives a briefly background introduction of posttranslational modification research, including the strategy of identification for posttranslational modification and the development of acetylation. This study consisted of two parts, which were introduced respectively in chapter 2 and 3 in detail. In chapter 2, we used two kinds of mass spectrometers including ESI-MS(LC-IT-TOF and LTQ-Orbitrap) and MALDI-MS (4700 Proteomics Analyzer and QIT MS) to identify a standard acetylated protein BSA-ac, for the reference of the identification of acetylated proteins in mouse liver. It was demonstrated that, LTQ-Orbitrap (LC-ESI-MS) and 4700 proteomic analyzer (MALDI-TOF-TOF) can be better used in the identification of acetylation compared with the other two spectrometers. Additionally, these two methods were complementary to each other in analyzing acetylated proteins. In chapter 3, we designed two experimental proposals for the identification of acetylated proteins of mouse liver. The first, we used 2DE gel for the separation of liver proteins, 2D western-blotting for the detection of the acetylated proteins, we chose the spots that showed positive in the 2D western-blotting and analyzed them using 4700 Proteomic Analyzer. We identified 6 lysine acetyalted proteins and 8 lysine acetylated sites. The second, we used immunoprecipition to enrich the acetylated proteins, and analyzed them using LTQ-Orbitrap. We identified 91 lysine acetyalted proteins and 216 lysine acetylated sites. A lot of acetylated proteins that we identified were not reported before. The identified lysine acetylated proteins were classified bioinformatically respective to their function, biological process and intracellular localization. Additionally, we used the Q-star mass spectrometer (ESI) to analyze threonine and serine acetylated proteins, and identified 18 threonine acetylated proteins with 21 threonine-acetylated sites and 25 serine acetylated proteins with 30 serine-acetylated sites. The chapter 4 summarized the development of acetyalation, and gave a prospect for this study.The study firstly combined two kinds of mass spectrometry techniques for the large scale identification of mouse liver, which is an ideal model sample for human liver. The data sets offer a rich source for further characterization of the contribution of this modification to cellular physiology and human diseases.