Molecular signatures of rheumatoid arthritis characterized by transcriptome and proteome analysis

Abstract

Our research is dedicated to the molecular analysis of autoimmune diseases with a strong focus on rheumatoid arthritis (RA). In order to detect disease-relevant processes and biomarkers in patients suffering from RA, we searched for conspicuous differences in the abundance of soluble proteins between synovial fluids (SF) and plasmas of patients. Detailed analysis of two-dimensional gel electrophoresis-separated protein spots by means of MALDI-MS and MALDI-QIT-ToF-MSn sequencing [1,2] revealed that the haptoglobin α-chain is present in both body fluids in at least four variants. This result suggests that specific post-translational modification processes occur in SF that could be playing an important role in the inflammatory degradation process of the joint. We therefore analyzed pannus tissue from RA patients and compared the obtained protein pattern with that from tissues derived from patients with osteoarthritis (OA) first by RNA microarray-analysis (Affymetrix) [3,4]. Our combined approach started with screening the human genome for protease genes. There are 590 proteases encoded by the human genome, 397 of which are found on the Affymetrix chips, represented by 687 unique probe sets. In addition, from the 159 protease inhibitors in the genome, 106 are presented on the chips by 164 unique probe sets. Interestingly, only approximately 330 protease probe sets and approximately 70 inhibitor probe sets yielded in so-called 'present calls'. A comparison of the RNA abundances between RA and OA showed that approximately 40 protease and 16 inhibitor gene products could be identified as differentially expressed. One of them was Cathepsin B, which was found approximately twofold more expressed in the pannus (RA) than in synovial tissue (OA) at the RNA level. Parallel proteomics investigations showed over 800 protein spots in the gels from tissue materials. Approximately 250 of the protein spots were analyzed by mass spectrometry, resulting in the identification of approximately 180 unique proteins. Among the identified proteins 12 belonged to the group of proteases. Again, Cathepsin B was found significantly upregulated. In addition, the small calcium binding protein S100A9 (MRP14) was identified as a discriminatory marker protein in SF by global proteomic analysis [5,6]. These results were confirmed by ELISA and it was found that plasma levels of the S100A8/A9 heterocomplex are correlating well with levels in SF; hence determination of plasma levels is applicable for distinguishing RA patients from patients with other inflammatory joint diseases as well as from OA patients and from controls. Initial studies on RA samples indicate that plasma levels of the S100A8/A9 heterocomplex might become a useful marker in monitoring anti-tumor necrosis factor alpha therapy responses.