Comparative Proteomic Study Of Human Peripheral Blood Mononuclear Cells In Systemic Lupus Erythematosus Patients By Mutiple Labeling Of Isobaric Tags For Relative And Absolute Quantitation And Tandem Mass Spectrometry

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disorder with unpredictable outcome and a long clinical course characterized by periods of both active disease and remission. The pathogenesis of SLE is incompletely understood. In present study, highly throughput quantitative proteomic technology-isobaric tagging for relative and absolute protein quantification (iTRAQ) combined with tandem mass spectrometry has been firstly applied to identify and quantify total proteins in peripheral blood mononuclear cells (PBMC) of SLE patients . By this way, we attempt to establish SLE-specific proteome profiles and find new important factors involved in the lymphocytic regulatory pathway in SLE, which would help better understand the pathogenesis of SLE and develop a new way for diagnosis and treatment of SLE. Initial mass spectrometry based proteomic analysis revealed that serine-threonine kinase receptor-associated protein (STRAP) significantly decreased in active SLE. The under-expression of STRAP was further confirmed by immunoblot detection and its correlation with SLE progression was analysed. STRAP may be of clinical significance as a potential state-specific indicator in active SLE.PARTâ… THE REPRODUCIBILITY AND ACCURACY OF ITRAQ MULTIPLE LABELING TECHNIQUE IN PROTEIN QUANTITATIVE ANALYSIS OF HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLSObjective To establish and optimize proteomic research platform using isobaric tags for relative and absolute quantitation (iTRAQ) so as to facilitate further proteomic research of human peripheral blood mononuclear cells. Methods Human peripheral blood mononuclear cells (PBMC) were collected. After protein extraction and trypsin digestion, samples were labeled with iTRAQ reagents and then subjected to mass spectrometry. In triplicates, thirty precursors were randomly selected and detected. Results as a result, 26, 28 and 29 peptides were respectively tagged with iTRAQ reporter ions. The labeling efficiencies ranged between 86.7% ~ 96.7%, with no significant difference among groups (P > 0.05). The coefficient of variance for the relative ratios of peptides from different proteins was ranged from 7.6% to 25.5% and there were no significant differences across groups either (P > 0.05). The coefficient of variance for the relative ratios of different peptides from the same protein was varied from 9.3% to 19.1% and the differences across groups were not significant (P > 0.05). Conclusions The labeling of iTRAQ combined with tandem mass spectrometry in PBMC is successful with favourable reproducibility and accuracy, which lays a foundation for further proteomic study of human peripheral blood mononuclear cells in autoimmune disorders.PARTâ…¡COMPARATIVE PROTEOME ANALYSIS OF PERIPHERAL BLOOD MONONUCLEAR CELLS IN SYSTEMIC LUPUS ERYTHEMATOSUS WITH ITRAQ MULTIPLE LABELING TECHNOLOGYAim To identify and quantify total proteins in peripheral blood mononuclear cells of systemic lupus erythematosus patients with quantitative proteomic technology, and to find differential expression proteome profiles of SLE. Methods Four-plex isobaric tags for relative and absolute quantification coupled with multiple chromatographic fractionation and tandem mass spectrometry were used to analyse total proteins in PBMC from patients with stable SLE, active SLE, rheumatoid arthritis (RA) and healthy controls. Proteins were identified by database searching with peptide map fingerprinting (PMF) and their expression differences were compared. Results More than four hundred proteins were identified. Compared to healthy controls, 44 proteins were discovered to be differentially expressed by more than two folds in stable SLE and active SLE, and among them 9 proteins were up-regulated and 35 proteins down-regulated. Compared to RA group, a total of 52 proteins displayed two or more fold changes in stable SLE and active SLE, including 19 up-regulated proteins and 33 down-regulated ones; the up- and down-regulated proteins between active SLE and stable SLE were 17 and 13, respectively. Conclusions Quantitative proteomic technology is efficiently applicable for protein identification and relative quantitation of human peripheral blood mononuclear cells. Differentially expressed proteome profiles of SLE patients are determined. Further investigation on the molecular mechanism of the involved proteins may help better understand the pathogenesis of SLE and develop a new way for diagnosis and treatment of SLE. Objective To analyze the expression levels of serine-threonine kinase receptor-associated protein (STRAP) in peripheral blood mononuclear cells (PBMC) of systemic lupus erythematosus patients and to investigate the possible application of STRAP test in SLE pathologic state evaluation. Methods PBMC from stable, active SLE patients and healthy controls were collected, respectively. STRAP was first identified and quantified with isobaric tagging for relative and absolute protein quantification combined with tandem mass spectrometry, and then confirmed by Western blotting in larger independent sample size. Results Initial mass spectrometry based proteomic analysis revealed that STRAP significantly decreased in active SLE compared with healthy controls (the relative ratio was 0.2906). The under-expression of STRAP was further confirmed by immunoblot detection(P < 0.05). Clinical data analysis disclosed that the expression levels of STRAP in SLE inversely correlated with SLE disease activity index (r = -0.607, P < 0.05). Conclusions STRAP may be involved in the pathogenic process of SLE, and be of clinical significance as a potential condition-specific indicator of active SLE.