Peptidomic Profiling of Plasma for AMD Biomarkers

Abstract

Purpose: : To develop a blood test for age–related macular degeneration (AMD) that will allow identification of those at risk prior to clinical evidence of the disease.

Methods: : Blood was collected from clinically documented AMD and age–matched normal, healthy donors at the Cole Eye Institute, Cleveland Clinic Foundation and Louis Stokes Cleveland VA Medical Center. Plasma carboxyethylpyrrole (CEP) immunoreactivity and CEP autoantibody titer were determined by ELISA. Plasma was prefractionated on reversed phase or anti–CEP antibody coated magnetic beads then analyzed by high resolution MALDI TOF mass spectrometry to detect peptides. Logistic regression modeling of ELISA data for the c–statistic and odds ratio was performed with SAS/STAT software. Cluster analysis and cross validation of peptidomic patterns was performed with GeneSpring software.

Results: : Progress in ELISA analyses of plasma from AMD and normal control donors has extended preliminary results (2003 J Biol Chem 278, 42027) and confirmed that AMD donors (n = 518) exhibit overall higher mean levels of CEP immunoreactivity and autoantibody titer relative to age–matched normal donors (n = 218), including early stage AREDS AMD category 2 donors. MALDI TOF mass spectrometric analyses of CEP immunoaffinity fractionated plasma (90 AMD and 80 normal plasma) has allowed correct prediction of 94% as either AMD or normal based on peptidomic profiles. Structural characterization is underway of peptides that appear to be significantly different in AMD and normal plasma.

Conclusions: : Plasma CEP immunoreactivity and autoantibody titer are typically elevated in AMD patients. Plasma peptidomic patterns may provide a method for early identification of individuals susceptible to developing AMD, before retinal degeneration.

Keywords: age-related macular degeneration • oxidation/oxidative or free radical damage • proteomics