Primary biliary cirrhosis (PBC) is an autoimmune chronic liver disease characterized by autoantibodies to mitochondria. The autoantigens are highly conserved, intracytoplasmic enzymes that have been identified as components, of the 2-oxo-dehydrogenase pathway. The major autoantigen is the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Our laboratory has previously characterized the mitochondrial autoantibodies, cloned and performed epitope mapping of the autoantigens, developed newer diagnostic assays using recombinant fragments, generated mouse monoclonal and human combinatorial antibodies to PDC-E2, and also reported establishment and repertoire analysis of T cell cloned lines. Using many of these reagents, we have demonstrated that patients with PBC, but not controls, have either PDC-E2 or a cross-reacting molecule on the apical or luminal side of biliary epithelium. The identity of this molecule is unknown, but its appearance is the first known marker of PBC. To perform a complete analysis of the molecular determinants of PBC, and to identify unique biomolecules associated with biliary epithelium, we will take advantage of our development of, and experience with, surface-enhanced laser desorption/ionization (SELDI) to evaluate molecular markers for PBC. This technique has allowed us to accomplish """"""""nanoscale"""""""" biopolymer detection (i.e., protein discovery) and structural characterization, referred to """"""""affinity mass spectrometry"""""""" (AMS). In particular, we can actively dock on a mass spectrometer probe element surface extremely small quantities (< fmole) of specific macromolecules from unfractionated sources. Our new probes permit analyte quantitation and biopolymer sequence analysis by laser induced desorption-ionization time-of-flight (TOF) mass spectrometry (MS). In fact, we can perform molecular analysis on a scale previously considered impossible using only frozen sections of hepatic tissues or isolated biliary epithelial cells. We will use SELDI-TOF MS in the AMS mode and probes designed for surface enhance affinity capture (SEAC) to develop a molecular mass difference map and identify by in situ sequence analysis those biomolecules uniquely associated with PBC bile duct epithelial cells. We will also use SEAC probes with monoclonal/ combinatorial antibodies to capture/identify specific autoantigens in liver and bile duct epithelial cells. The data obtained for this proposal will be valuable not only for our understanding of PBC, but also for the use of similar technology in other autoimmune diseases.
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