Proteomic Research In Membrane Microdomains Protein From Hepatocytes During Liver Regeneration

Liver has a remarkable capacity to regenerate after hepatectomy. The research of liver regeneration may provide a theoretical basis for the treatment of liver injury and liver transplantation. Lipid rafts and caveolae microdomain, as specialized regions of plasma membrane, play important roles in the endocytosis, pinocytosis, signal transduction and the transportation of cholesterol. In the process of liver regeneration, protein component of lipid raft in the microdomains was changed based on the internal regulating signal system. Analysis the variation of lipid raft proteins composition may be helpful to explore the signal pathway involved in liver regeneration. In this study, a model of2/3partial hepatectomy combined with sucrose density centrifugation were used to purify the plasma membrane from the rats in sham operation control group (0h) and2/3partial hepatectomy (48h) individually. The membrane proteins were separated by SDS-polyacrylamide gel electrophoresis and identified by ESI-Q-TOF MS/MS analysis. Among the differential expressed proteins,17proteins were up-regulated and23proteins were down-regulated. Those proteins involved in cell proliferation, programmed cell death, apoptosis and angiogenesis signaling pathway.Flotillin-1, as a known lipid rafts specific protein, can be used as biomarkers for quantitative analysis to complex sample. In this study,2peptide sequence were selected from flotillin-1and synthesized as standard, which were labeling by stable isotope (SIL), to verified the complex sample. Synthetic peptides were high-purity with a definite sequence. The new method is based on combining multi reaction monitoring-mass spectrometry/mass spectrometry (MRM-MS/MS) with18O labeling analysis. By obtained the peak area ratio and knowing the quality of synthetic peptides, flotillin-1in sample can be quantificated by data analysis. In our result, the synthetic peptide sequence can be well fragmented and identified by MS/MS analysis, and the complex sample can differ by4Da to standard. Protein validation method based on mass spectrum give a new insight to protein identification. Our study might provide clues to the research of microdomains proteomics and laid foundations for subsequent absolute quantitative proteomic approach.