Protein Post-Translational Modification Research Of C57 Mouse Liver Endoplasmic Reticulum

Endoplasmic Reticulum (ER) is an important cell organelle which is correlated to the quantity changes of protein due to normal physical change or stimulus of environment. The proper function of ER in cell is indispensable. Most lipids and proteins, which include the one secreted and the one located in the apparatus such as plasma membrane, mitochondrion, and lysosome, are synthesized in ER. Moreover, ER is also involved in the process of cell detoxification and Calcium ion deposition.Phosphorylation and Glycosylation represent the most important and widespread role among more 200 types of protein post-translational modifications. Glycosylation of a protein changes its physical and chemical characteristics greatly, such as mass and solubility. It also influences proteins function at same time. Glycoprotein accounts for 50% of proteins in cell. But due to its inherent heterogeneity, researching on glycoprotein is also a tough challenge to proteomics scientists. Protein phosphorylation plays a key role in regulating physiological courses and is involved in most physiological and pathological course in life, such as metabolism, cell growth, and carcinogenesis. Therefore, more and more proteome researche has been focused on the field of protein phosphorylation. Systematical studies on phosphorylation proteome of living organism will help us to elucidate the physiological and pathological mechanism involved by phosphorylation. But the characteristics of phosphoprotein, such as low stoichiometry and heterogeneous complexity, made studies on this field a tremendous challenge.C57BL/6J mouse is a major model organism due to its unparalleled genetic and biochemical tractability. 8-9 weeks old male C57BL/6J mice were used in this study to research Glycosylation and Phosphorylation of ER protein.In the first part of the paper, the strategy of ER N-glycoproteins research based on ConA affinity chromatography was established. The lectin ConA was used in the enrichment of N-glycoproteins because of its high affinity. First, we optimized the ConA affinity chromatography in order to capture the low abundance N-glycoproteins in complex sample. Then, we extracted the ER of mouse liver by sucrose density gradient centrifugation methods, and captured the N-glycoproteins based on ConA affinity chromatography. Finally, the N-glycoproteins were identified and N-glycosylation site were confirmed by linear ion trap-Fourier transform ion cyclotron resonance-mass spectrometry (LTQ-FTICR MS) with high mass accuracy. Totally 45 mice were utilized and separated to three groups. Three replicated LTQ-FTICR experiments were conducted for each mice group. A total of 322 glycosylation sites on 308 unique glycopeptides belonging to 212 unique N-glycoproteins are identified from 9 batches of raw data, of which, 131 proteins are known N-glycoproteins in Swissprot database.In the second part, the phosphoprotein identification strategy based on SCX-LC-MS/MS was optimized by using standard phosphoproteins and mitochondrion proteins, and then the strategy was used to characterize the phosphoproteome of the mouse liver ER. In total 249 phosphorylation sites and 164 phosphorylated peptides were identified from 161 phosphoproteins in the mouse liver ER. Compared to Swissprot database, 125 phosphoproteins are novel. Most of them play an essential function closely associated with the physiological and pathological mechanism in mouse liver ER.We established the strategy to research N-glycoproteome and phosphoproteome of mouse liver ER based on ConA lectin chromatography and SCX-LC-MS/MS method. The profile of N-glycoprotein and phosphoprotein of mouse liver ER were established at first time. These results would allow further insight into the function of ER of mouse liver.