The Research Of Quantiative Protemics In The Patients With Lupus Nephritis

Objective:Isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) were used to screen the proteins differential expression in the blood serum of Systemic Lupus Erythematosu with and without lupus nephritis(LN and SLE). Thus, the finding may afford new angle and thought for LN early diagnosis and condition monitoring through the serum quantitative proteomic.Methods:Firstly, the patients were divided into three groups, namely LN, SLE and control groups. Next, the groups were renamed for LN-low, SLE-low and N-low after using the Agilent multiple affinity removal LC column-Human 14(MARS) to remove 14 species of high abundance proteins in all serum samples and then concentrate the low abundance proteins. All the differential proteins of SLE and LN that screened by iTRAQ labeling combined with mass spectrometric analysis were further analyzed by Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) to explore the bioinformatics. Western blot analysis was applied to validate part of the proteins(APOE、CIQC、SAA1) to confirm the iTRAQ results. Further study of SAA1 was conducted by ELISA analysis, and the result was analyzed by SPSS21.0.Result:1. SDS-PAGE electrophoresis and Quantity One application were applied to verify the removal efficiency of high abundant proteins, and our results indicated that most of the high abundant proteins were removed.2. The iTRAQ labeling combined with 2D-LC-MS/MS proteomic analysis identified 302 non-redundant proteins (95% confidence interval). After setting the screening criteria for differentially expressed proteins,243 proteins were included. The screening criteria were ProtScore (unused)>1.5 and P<0.05, with its Ratio>1.2 means expression of the proteins was up-regulated, while its Ratio<0.8 means expression the protein was down-regulated. When compared with the normal control population,235 and 243 differentially expressed proteins were found in LN and SLE group, respectively. Among them,233 up-regulated with two down-regulated proteins were in LN group, and three up-regulated with 240 down-regulated proteins were in SLE group. When compared the SLE group with LN group, there were 243 differentially expressed proteins, and all of them were up-regulated expression.3. According to the biological information and IPA analyses of the differentially expressed proteins, our results indicated that the proteins mainly located outside the cell and play roles in the inflammatory reaction, and the signaling pathways these proteins involved mainly consist of L activation FXR/RXR activation and complement system. A receiver operating characteristic curve (ROC) was used to evaluate the SAA1 expression in serum of SLE and LN patients, the area under the ROC curve was 0.843, P=0.001 for the detection of LN. And the Cut-off value of SAA1 was 1.555μg/mL, with its sensitivity and specificity was 70.5% and 85.7%, respectivelyConclusion:1.243 differentially expressed proteins were found between the LN and SLE by iTRAQ and 2D-LC-MS/MS. And the serum SAA1 was proved as a potential biomarker for the detection of early LN.2. Many ingenuity pathways play important role in SLE and LN, such as abnormal immunity, acute inflammatory responst and abnormal blood lipid and so on. The body’s inflammatory reaction may be the main factors contributing to the development of SLE and LN.