Evaluation of a Plasma Biomarker for Advanced Age-Related Macular Degeneration Using Nanotrap® Particle-Enriched Proteomics Kirk Hansen (Presenter) University of Colorado Presenter Bio: Dr. Hansen is an Associate Professor in the Department of Biochemistry and Molecular Genetics at University of Colorado Denver Medical Campus. He is also Director of the Cancer Center Shared Proteomics Facility and co-founder of Omix Technologies. He has extensive experience in the identification and characterization of low-level peptide and protein samples (18 yrs). WIth a focus on differential expression strategies for the characterization of complex protein mixtures from mammalian tissue, cell-lines and biofluids.

Authors: Kirk C. Hansen(1,2), Erica Porter (3), Robbie Barbero (3), Ben Lepene (3), Alan G Palestine(4), Anne M Lynch(4), Brandie D Wagner(4), Jennifer L Patnaik(4), Marc T Mathias(4), Frank S Siringo(4), Ram Nagaraj(4), Naresh Mandava(4), Monika Dzieciatkowska (2)
(1) University of Colorado, School of Medicine, Department of Biochemistry and Molecular Genetics. (2) Proteomics Shared Resource, Aurora, CO, USA. (3) Ceres Nanosciences, Inc., Manassas, VA, USA. (4) University of Colorado, School of Medicine, Department of Ophthalmology.

An aptamer-based technology was used to discover biomarkers of age-related macular degeneration. Vinculin, which is a cytoplasmic cytoskeletal protein involved in cell adhesion and mobility and which is found in low concentration in plasma, was identified as a promising candidate biomarker. An orthogonal liquid chromatography approach with Nanotrap® particles, which are engineered hydrogel particles functionalized with chemical affinity baits, was utilized to validate this finding. Direct measurements of tryptic peptides generate more than 100 candidates that exhibited improved statistical significance between case and controls.

Introduction

Age-related macular degeneration (AMD) is a progressive degenerative disease of the macula usually found in patients who are over 60 years of age. AMD accounts for approximately 9% of blindness worldwide. AMD is distinguished by two advanced forms of the disease based on choroidal neovascularization (NV) and atrophy of the RPE with overlying photoreceptor cell atrophy (geographical atrophy - GA). There is currently a great need for discovery of biomarkers for the diagnosis, prognosis, and to inform the treatment of AMD.

Methods

Thirty samples were used from the AMD Registry. Plasma from 10 age-matched patients with bilateral geographic atrophy AMD (GA), neovascular AMD (NV) and 10 controls without retinal disease that had previously been analyzed for 4001 proteins using the SOMAscan® platform were utilized. Ceres Nanosciences’ Nanotrap® particles were used to enrich and digest proteins from 50 microliters of plasma from the same samples before LC/MS analysis. Statistical approaches were used for validation of vinculin, data mining, and identification of improved candidate markers of disease.

Results

The Nanotrap® particle enrichment and digestion approach allowed for over a 2-fold increase in the number of proteins identified from plasma when compared to a non-enrichment approach. The mass spectrometry approach revealed vinculin was 1.66 fold elevated in GA ( p<0.01) and 1.84 fold elevated in NV (p<0.006) compared with controls. This was consistent with vinculin levels as determined by the aptamer approach; GA (p<0.13) and for NV (p<0.01). The Nanotrap® particle-LC/MS approach identified more than 100 proteins that exhibited more significant changes between disease state and controls then vinculin.

Conclusions & Discussion

Using an independent analytic technique that utilized Nanotrap® particles to drastically improve plasma proteome coverage, we have confirmed that plasma vinculin is elevated in advanced AMD compared with controls. This confirms a previous report in NV patients by Kim, et al in 2014, and our work with the SOMAscan® platform. In addition, a large number of novel candidate biomarkers were identified with multiple unique peptide measurements.