A project beginning half-way through this year was started by postdoctoral fellow Leticia Cano and attempts to identify biomarkers in autoimmune disease using protein profiling in combination with mass spectrometry. Proteins isolated from tissue and immune complexes will be used as a source of biomarkers. Collaborations have been established with several clinical groups involved in Sjogren's disease, systemic lupus erythematosus and inflammatory myositis (G. Illei (NICDR), F. Miller (NEIHS) and E. Rushing (AFIP). We have received OHSR approval to obtain human samples from the Cooperative Human Tissue Network (CHTN) and AFIP (Rushing). Tissues obtained from healthy controls and disease controls will also be examined to ensure that we do not identify markers of inflammation. New ways to separate and profile proteins prior to LC/MS/MS analysis are being investigated(chromatofocusing, ion exchange, salt precipitations and affinity columns). A Beckman PF2D system for 2D-HPLC separation of intact proteins has been acquired and will be examined for fractionation of immune complexes and proteins isolated from human tissue targeted in autoimune disease. Ammonium sulfate precipitation with a countercurrent precipitation chromatography system (Y. Ito, NHLBI) was examined as a means of isolating immune complexes; however, many other proteins precipitated with the complexes. We profiled the fraction that solubilized after disrupting the complexes with NaOH. For Sjogren's syndrome, two peaks were distinguished from the healthy control. LC/MS/MS analysis of these fractions identified complement C4A and apolipoprotein A-IV in the large peak. The smaller peak contained apolipoprotein A-I, complement C3, phosphoglycerate kinase 1 and complement factor H. Apolipoproteins are markers of inflammation, and this data merits disease-specific confirmation. Profiling of the samples from patients with inflammatory myositis was too complex and did not yield a disease-specific pattern. We also used a column from ProGen Biologics to isolate circulating immune complexes. LC/MS/MS analysis of the antigens eluted from the complexes identified the following proteins after profiling: SLE: coagulation factor V, glyceraldehyde-3-phosphate dehydrogenase, histidine-rich glycoprotein, insulin-like growth factor 2 receptor, profilin and tenascin, and dermatomyositis: many proteins including alpha-2-HS-glycoprotein, moesin, transgelin 2, tyrosine 3/tryptophan 5-monooxygenase actionation protein, and myosin regulatory light chain 9. However these protein hits have not been verified by other techniques and we are in the methods development/discovery phase of the project.
