| Objective: Differential proteins in blood and urine exosomes from LN patients and Normal Control(NC)were analyzed using whole protein TMT labeling quantitative proteomics in attempt to find new diagnostic biomarkers for LN.Methods: Serum and urine exosomes from LN and NC patients were extracted using the kit method,identified by exosome characterization by electron microscopy,particle size,and Western Blot,and the extracted exosomes were subjected to protein extraction,peptide digestion,TMT labeling,chromatographic classification,LC-MS /MS data acquisition,and database search.Significantly different proteins were screened by FC > 1.2-fold and P < 0.05,and the numbers of up-and down-regulated proteins between LN and NC serum and urine were obtained for each comparison group.The resulting significantly different proteins were subsequently analyzed bioinformatically,and the analysis mainly included identification analysis,expression difference analysis,and functional analysis.Clinical data and laboratory examination of LN patients were collected: such as sex,age,course of disease,fever,butterfly erythema,photoallergy,hair loss,mucous ulcer,arthritis,serositis,vasculitis,cerebrovascular accident,nervous system abnormality,blood routine,routine urine,creatinine,24-hour urine protein quantification,serum albumin,complement C3,complement C4,autoimmune antibody(Anti ds DNA antibody,anti SM antibody,anti SSA/ro60 kd antibody,anti SSA/ro52 kd antibody,etc.).SPSS 25.0 software was used to analyze the experimental and clinical data,and all the data were analyzed in normal distribution.The comparison between two groups of normal distribution data is conducted by two groups of independent sample t test,and non-normal distribution data is compared by rank sum test.Pearson correlation coefficient analysis is used for correlation analysis between two groups of normal distribution data,and Spearman rank correlation coefficient is used for correlation analysis of partial distribution data,logistic regression analysis is used for the correlation analysis of the two classification data,all statistics The analysis was bilateral,P <0.05,the difference was statistically significant.Results:(1)The results of electron microscopy showed that the exosomes were spherical vesicles with uniform size and intact morphology;the results of particle size-NTA assay showed that the diameter of exosomes was 30-200 nm;Western-blot protein verification showed that the LN group exosomes were positive for CD63 and TSG101.(2)A total of 677 proteins were identified in serum exosomes,and 672 of them could be quantified.Fold Change(FC)> 1.2-fold and P < 0.05 was the criterion,yielding 106 significantly different proteins in the comparison group.Based on the above data,subcellular localization of significant difference proteins was carried out.Most of the proteins were mainly distributed in extracellular,cytoskeleton,nucleus and plasma membrane.In GO analysis,in terms of cellular components,serum exosomes differential proteins were mainly concentrated in extracellular matrix,basal lamina,platelet dense granule lumen and other components;in terms of molecular functions,serum exosomes differential proteins were mainly concentrated in ion channel inhibitor activity,channel regulator activity,antigen binding and other functions;in terms of biological processes,serum exosomes differential proteins were mainly involved in In biological processes,serum exosomes differential proteins are mainly involved in receptor-mediated endocytosis,protein activation cascade,negative regulation of humoral immune response,etc.Analysis of KEGG signaling pathway showed that serum exosomes differential proteins are mainly involved in asthma,Intestinal immune network for Ig A production,phospholipase D signaling pathway,rheumatoid arthritis and other pathways.A total of 3414 proteins were identified in urine exosomes,3355 of which were quantifiable,yielding 157 significantly different proteins in the comparison group,using FC > 1.2-fold and P < 0.05 as the criterion.Based on the above data,subcellular organelle localization of significantly different proteins,most of which are mainly distributed in the extracellular,cytoplasmic,and nuclear regions.In GO analysis,in terms of cellular composition,urinary exosomes differential proteins were mainly in the extracellular region,extracellular space,extracellular vesicles and other components;in terms of molecular function,urinary exosomes differential proteins mainly in peptidase regulator activity,immunoglobulin receptor binding,serine-type endopeptidase inhibitor activity and other functions;in biological processes,urinary exosomes differential proteins are mainly involved in immune responses,positive regulation of the establishment of protein localization to telomeres,and regulation of protein localization on chromosomes and other processes.KEGG signaling pathway analysis revealed that urinary exosomes differential proteins are mainly involved in lysosomal,PI3K-Akt signaling pathway,tight junctions,pathogenic Escherichia coli infection,and apoptosis.(3)Further analysis of the correlation between serum exosome SVEP1 and PCSK9 protein expression levels and laboratory indices in LN patients showed that serum exosome SVEP1 expression was negatively correlated with glomerular filtration rate in LN patients(r =-0.738,P = 0.023).the correlation between the expression levels of urinary exosomes proteins ENG and GOT1 and laboratory indices in LN patients.The results showed that the expression of ENG in urinary exosomes of LN patients was significantly and negatively correlated with the levels of C3(r =-0.8,P = 0.01)and C4(r =-0.745,P = 0.021).In the differential analysis of SVEP1 and PCSK9 protein expression levels in the serum group,the expression of PCSK9 in the ds-DNA antibody-positive group was significantly lower than that in the negative group(P < 0.05);in the differential analysis of ENG and GOT1 protein expression levels in the urine group,the expression of ENG in the C3 and C4-reduced group was significantly higher than that in the non-reduced group(P < 0.05).Conclusion: The proteomics in two groups of exosomes,serum group and urine group,were analyzed separately using LC-MS/MS mass spectrometry proteomics.By identifying the differential expression of serum and urine exosomes proteins in LN patients and healthy controls,a total of 106 significantly different proteins were screened in the serum group and157 significantly different proteins were screened in the urine group,with a total of 13co-expressed proteins in both groups.Based on the differential analysis,correlation analysis of laboratory tests in LN patients and combined with relevant literature reports,we found that SVEP1,ENG,PCSK9 and GOT1 associated with inflammatory response could be used as diagnostic biomarkers for LN and have the potential to be biomarkers for assessing LN disease activity.The next step is to validate these biomarkers in large samples,and it is scientifically important to study their value in the pathogenesis of LN and in the diagnosis,efficacy and prognosis. |
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