Identification of Carboxyethylpyrrole-Modified Proteins in AMD Plasma

Abstract

Purpose: : Previous work has shown that carboxyethylpyrrole (CEP) adducts, uniquely derived from the oxidation of docosahexaenoate-containing lipids, are elevated in Bruch’s membrane and plasma from donors with age related macular degeneration (AMD). We have sought the identity of CEP-modified plasma proteins for AMD biomarker discovery and for insights to mechanisms in AMD pathogenesis.

Methods: : Immunoprecipitation (IP) of CEP-modified proteins in AMD plasma (1 ml) from two patients exhibiting high CEP immunoreactivity (~120 pmol/ml) was performed with anti-CEP IgG monoclonal antibody covalently linked to protein G agarose beads. IP products were analyzed by Western blot and by LC MS/MS following SDS-PAGE with gel bands excised and digested in situ with trypsin. Peptides in AMD (n = 30) and control (n = 30) plasma were captured on anti-CEP antibody beads, analyzed by MALDI TOF MS and statistically significant differences between AMD and control plasma peptide patterns determined (GeneSpring). Peptides immunocaptured from AMD plasma were pooled and analyzed by LC MS/MS. Proteins were identified using the Mascot search engine and the Swiss-Protein and NCBI sequence databases. CEP modifications sites were determined by manual examination of MS/MS spectra. The identified CEP- proteins were subjected to bioinformatic pathway analysis (MetaCore).

Results: : Western analysis revealed many CEP-modified proteins in AMD plasma. Following IP of intact proteins, CEP-modified lysyl residues were identified in 26 proteins, including among others, a B-cell receptor-associated protein, carbonic anhydrase, ubiquitin-protein ligase SMURF1, insulin-degrading enzyme, complement C1q subcomponent C, synaptotagmin-10, and 2 protein kinase C isotypes. Immunocaptured peptides provided the identity of 5 CEP-modified proteins in AMD plasma, and included a peptide from a WW domain-containing oxidoreductase that was significantly different from peptides in control plasma (p = 0.004).

Conclusions: : We have identified a few of the CEP-modified peptides and proteins in AMD plasma. These proteins are associated with multiple metabolic pathways such as immune inflammation, ionic homeostasis, cell signalling and communication. The present technology offers promise for biomarker discovery and insights to the role of oxidative modifications in AMD pathology.

Keywords: age-related macular degeneration • oxidation/oxidative or free radical damage • protein modifications-post translational