ABSTRACT
Objective To explore the related protein which lead to rheumatoid arthritis (RA) and to find different proteins associated with active RA by comparing the expression levels of proteins in the peripheral blood mononuclear cells (PBMCs) of healthy individuals to patients with rheumatoid arthritis using a proteomics approach. Methods Samples of peripheral blood were collected from 9 patients diagnosed as active RA and 9 healthy individuals. PBMCs were isolated from blood using lymphoeytes separation medium. The total protein was extracted from the peripheral blood mononuclear cells. The total protein from either RA patients or normal controls was prepared by means of immobilized pH gradient based on two-dimensional gel eleetrophoresis. After Coomassie brilliant blue G250 staining, gel-image analysis was performed by using PDQuest.The differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALD I-TOF-MS). Then APOA-I was validated by RT-PCR. Results 2-DE patterns of PBMCs from controls and RA patients were presented. The results showed that the average number of protein spots was 556 and 579 respectively, and the corresponding average matching rate was 89.4％ and 88.5％ respectively. Gel-image analysis revealed that there were 23 differential protein spots. Fourteen of total 18 differential protein spots were successfully identified by MALD I-TOF-MS, of which 8 proteins were upregulated such as actin beta, fibrinogen beta chain, ApoA-I ; and 6 proteins such as peroxiredoxin-2, glu-tathione S-transferase omega 1 were down-regulated when compared with controls. The result of ApoA-I by RT-PCR was consistent with the proteomics results. Conclusion Some differentially expressed proteins are observed in the PBMCs of patients with rheumatoid arthritis, which may play a potential role in the pathogenesis of RA.
ABSTRACT
Objective To investigate the comparative proteomes difference of PBMC between health adults and Parkinson Disease(PD) patients with Ganyang Huafeng syndrome and Xuexu Shengfeng syndrome.So to approach the essence of the two TCM syndromes from the protein expression level.Methods A series of methods,including immobilolized pH gradient two-dimensional polyacrylamide gel electrophpresis(2-DE),Coomassie Brilliant Blue staining,PDQuest 2-DE analysis software,peptide massfingerprint based on matrix-assisted laser desorption/ionization time of flying mass spectrometry(MALDI-TOF-MS) and SWISS-PROT database searching,were used to separate and indentify the proteome of PBMC between health adults and PD patients with Ganyang Huafeng syndrome and Xuexu Shengfeng syndrome.Results The good 2-DE pattern including resolution and reproducibility was obtained.After Coomassie Brilliant Blue staining,15 spots were incised and analyzed by MALDI-TOF-MS.The typic peptide masses were searched in the SWISS-PROT database by Mascot software.All proteins were preliminarily identified,which ralated to cytoskeleton,anti-oxidative stress,protein degradation,signal transduction and cell cycle,etc.Conclusions The well-resolved,reproducible 2-DE patterns of Ganyang Huafeng syndrome and Xuexu Shengfeng syndrome PD were established and revealed the protein expression difference of PBMC between the two syndromes of PD patients.This research is helpful to provide substance evidence in investigation of the essence of Ganyang Huafeng syndrome and Xuexu Shengfeng syndrome on protein level.