Immunoproteomic Analysis of Serum Antibody Complexes in Glaucoma Patients

Abstract

Purpose: : Glaucoma patients exhibit increased titers of serum antibodies to retina and optic nerve proteins. As part of studies exploring the regulation of immune response in glaucoma, this study aimed to identify disease-related antigens in serum IgG complexes.

Methods: : Serum samples were collected from 142 patients with high- or normal-pressure primary open-angle glaucoma or pseudoexfoliative glaucoma, and 70 control subjects without glaucoma. Serum protein complexes containing IgG were eluted using co-immunoprecipitation and spin-column purification, and were quantitatively analyzed by 2D-LC-MS/MS. For further validation, serum samples were also traditionally tested against human retinal proteins and recombinant proteins using Western blot analysis followed by 2D-LC-MS/MS for protein identification. Oxidized proteins containing reactive carbonyl groups were determined by 2D-oxyblot analysis and ELISA.

Results: : Quantitative 2D-LC-MS/MS analysis of the IgG-containing protein mixtures revealed a complex profile, which included proteins with potential relevance to glaucomatous retina as well as seemingly irrelevant proteins localized elsewhere. Proteins identified in serum IgG complexes included stress proteins, structural proteins, and mitochondrial enzymes that were previously presented as glaucoma-related antigens. Immunoproteomic analysis also identified many new antigens with high confidence (based on at least two identified peptide and a greater than 99.0% probability assigned by the Protein Prophet algorithm). These included different isoforms of neural cell adhesion molecule, integrin, annexin, dynein, CREB-binding protein, calmodulin-regulated spectrin, ubiquitin-protein ligase, and apoptosis-inducing factor 1. Glaucomatous samples exhibited greater levels of protein carbonyls (p<0.01), and most of the proteins identified in serum IgG complexes were oxidatively modified.

Conclusions: : Using a new immunoproteomic approach, this study identified various proteins in the serum IgG complexes sampled from different subtypes of glaucoma. Based on the findings that the antigenic proteins were oxidatively modified and included both neuronal and non-neuronal proteins, increased serum antibodies are likely a consequence of the disease and represent the immune system-driven tissue cleaning/healing process. Ongoing studies should determine whether increased serum antibodies also reflect the molecular imprint of disease-causing antigens.

Keywords: proteomics • immunomodulation/immunoregulation • neuroprotection