Part I: Comparison Of Collision-induced Dissociation And High-energy Collision Dissociation In Proteomics Analysis Part 2: Proteomic Analysis Of Urinary Differences In IgU Nephropathy Of Xinjiang Uygur And Han Nationality

Objective:To comprehensively compare the difference of collision induced dissociation (CID) and high energy collision dissociation (HCD) in proteome research.Methods:We analyzed the tryptic digests of bovine serum albumin (BSA) and cell lysates by LTQ-Velos Orbitrap. According to compared the number of peptides and proteins, we determined the difference of the two modes.Results:The HCD fragmentation identified more peptides and this mode showed higher MS/MS success rate and higher Mascot score in the tryptic digests of BSA. These results demonstrated that the HCD fragmentation had higher MS/MS spectra quality than CID. But in the tryptic digests of cell lysates, the CID fragmentation identified more MS/MS spectra, peptides and proteins, which indicated that it produced higher sensitivity. The HCD fragmentation showed higher MS/MS success rate and higher Mascot score distribution, demonstrating higher quality of MS/MS spectrum.Conclusion:Both CID and HCD modes could be used for large-scale proteomic analysis. The CID mode had higher sensitivity, and HCD mode could achieve higher quality of MS/MS spectrum.Objective:To screen differentially expressed proteins of IgA nephropathy (IgAN) by the proteomics analysis using isobaric tags for relative and absolute quantification (iTRAQ) combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) between Uygur ethnic and Han ethnic from Urumqi.Methods:We classified the urinary samples in 4 groups:Uygur IgAN, Uygur healthy control, Han IgAN and Han healthy control. After quantification and trypsin digestion of the protein extract from the 4 groups, the iTRAQ regents 114,115,116 and 117 were used to label the peptides of the 4 groups respectively. Then the mixture of the peptides was analyzed by 2D LC-MS/MS. The MS/MS data were searched against the Swissprot database using the Mascot software for peptide identification and the scaffold software for quantification. The fold change cutoff ratio>2 or ≤-2 was selected as differentially expressed proteins (P<0.05).Results:A total of 842 proteins were identified in this study. Based on the condition of screening differentially expressed proteins, comparing with Uygur healthy control group,168 proteins were significantly up-regulated and 56 were significantly down-regulated in Uygur IgAN group. In the comparison of the Han group, we found 96 significantly differentially expressed proteins (74 were significantly up-regulated and 22 were significantly down-regulated in Han IgAN group).Among the differentially expressed proteins,45 proteins were differentially expressed in both Han and Uygur IgAN groups. Both Han and Uygur IgAN groups had the activation of the immune system, and there was an abnormal metabolism of fatty acid, the extracellular matrix related protein was significantly increased, and the binding protein and intracellular protein was significantly decreased. The cell survival and cell viability were activated in Uygur IgAN, and the cell migration and cell motility were activated in Han IgAN. Coagulation system and complement system were activated in Uygur IgAN. The catalytic activity and the multicellular organisms process were activated in Han IgAN. Several differentially expressed proteins such as ADIPOQ, SERPINC1, ICAM-1, TGFBR1 and TIMP-1 were verified by Western-blot assay.Conclusion:The expression of differential protein in the urine can reflect pathophysiological changes in IgAN, and have important significance in the noninvasive diagnosis.