Abstract
Affinity mass spectrometry (AMS) was used to evaluate the structural diversity of the E2 component of pyruvate dehydrogenase complex (PDC) in normal and diseased liver cells, including those from patients with the autoimmune disease primary biliary cirrhosis (PBC). Two different antibodies to PDC-E2, the immunodominant mitochondrial autoantigen in patients with PBC, were used. AMS was performed directly on frozen liver sections and purified bile duct epithelial cells. Mass spectrometric signals associated with the molecular recognition of PBC-specific antigenic determinants were enhanced by an in situ enzyme-linked signal amplification process. Samples from patients with PBC gave strong positive signals for the antigen(s) recognized by the monoclonal antibody C355.1. Conversely, tissues from normal and disease controls showed only a minimal signal. AMS was used to identify specific antigenic determinants within the E2 component of PDC for comparison with unknown antigenic determinants observed by affinity capture with C355.1 monoclonal antibody from PBC samples. PDC components bound to C355.1 were mapped and identified by mass before dissociation from the E2 component. A similar approach was used to identify unknown antigenic determinants associated with PBC. We believe AMS may be an important new approach with wide application to the identification of molecules associated with a number of disease states.
Highlights
Affinity mass spectrometry (AMS) was used to evaluate the structural diversity of the E2 component of pyruvate dehydrogenase complex (PDC) in normal and diseased liver cells, including those from patients with the autoimmune disease primary biliary cirrhosis (PBC)
PBC is an autoimmune cholangitis characterized by the destruction of intra-hepatic bile ducts/bile duct epithelial cells and the presence of autoantibodies to mitochondrial antigens (AMA)
AMA were first identified nearly 30 years ago, it was not until the cloning of the mitochondrial autoantigens that the targets were identified as components of the 2-oxodehydrogenase pathway with the E2 component of the pyruvate dehydrogenase complex (PDC-E2) as the immunodominant autoantigen
Summary
Affinity mass spectrometry (AMS) was used to evaluate the structural diversity of the E2 component of pyruvate dehydrogenase complex (PDC) in normal and diseased liver cells, including those from patients with the autoimmune disease primary biliary cirrhosis (PBC). AMS was used to identify specific antigenic determinants within the E2 component of PDC for comparison with unknown antigenic determinants observed by affinity capture with C355.1 monoclonal antibody from PBC samples. Three different multimeric enzyme components are assembled, together with regulatory components, to generate the highly regulated function(s) of PDC. New approaches to the generation and characterization of useful antibodies have been explored Most recently, these included the use of surface plasmon resonance to conduct competitive (i.e. indirect) epitope mapping studies with libraries of monoclonal antibodies [7, 8]
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