Quantitative analysis of differential proteins in liver tissues of patients with non-alcoholic steatohepatitis using iTRAQ technology

Abstract

To screen differentially expressed proteins (DSPs) in the liver tissues of patients with nonalcoholic steatohepatitis (NASH) using proteomic technologies to identify potential therapeutic targets of NASH. Liver tissue specimens were obtained from 3 patients with pathologically confirmed NASH and 3 normal control subjects. The total proteins were extracted from the specimens, and iTRAQ reagent was used to label the peptides for liquid chromatography tandem mass spectrometry (LC-MS/MS) detection. The DSPs were identified by comparing the data against UniProt protein database using Mascot2.3.02 software and were annotated and enriched using GO database; KEGG database was used for enrichment of the pathways involving these proteins. Real-time fluorescent quantitative PCR (qPCR) was performed to detect the mRNA expressions of the significant DSPs in NASH. By the criteria that a DSP has >1.2 or < 0.8 fold difference between NASH group and the control group and with P < 0.05 as the threshold, a total of 648 significant DSPs in NASH were identified, including 246 up-regulated and 402 down-regulated proteins. GO functional enrichment analysis showed that the DSPs were involved mainly in small molecule metabolism, organic acid metabolism, oxygen acid metabolism and other biological processes, and were enriched in KEGG pathways including the metabolic pathways, complement coagulation cascades, and ribosomes. Among the 25 DEPs with a fold difference >2.0 or < 0.5 (P < 0.05), 6 proteins showed consistent results between qPCR verification and proteomic analysis, including 5 down-regulated proteins: Jumonji protein (JARID2), Lebasillinlike protein (LCA5L), synaptophysin 1 (SYN1) and collagen α-1 (XIII) chain (COL13A1), FYVE, RhoGEF and PH domain protein 5 (FGD5), and 1 upregulated protein glutathione S-transferase Mu 4 (GSTM4). We identified 648 DEPs inthe liver tissue of patients NASH using iTRAQ technology and bioinformatics methods, and among them JARID2, SYN1, COL13A1, FGD5, and GSTM4 may serve as the key target proteins of NASH.