Optical Nerve Fractionation for Proteomic Analysis

Abstract

Abstract: : Purpose: To evaluate the efficacy of solution state isoelectricfocusing (IEF) and 2D polyacrylamide gel electrophoresis (2D PAGE) for fractionation of optic nerve for proteomic analysis. Methods: Optic nerve was dissected from normal human eyes obtained through the Cleveland Eye Bank and the Eye Donor Program of the Foundation Fighting Blindness, Inc. Optic nerve protein extraction efficiency was evaluated in 7M urea and 2M thiourea containing either 6% C₇BzO, 3% ASB–14, 3% dodecylmaltoside (DM) or a proprietary detergent solution. Soluble protein was quantified by the Bradford and bicinchoninic assays. Following solution state IEF using the Multicompartment Electrolyzer (MCE, Proteome Systems) and 2D PAGE, gel spots were excised, digested in situ with trypsin and proteins identified by capillary LC MS/MS. Results: Optic nerve extracts from the four detergents produced similar 1D SDS–PAGE profiles, however, ASB–14 and dodecylmaltoside yielded slightly more soluble protein. Solution state IEF in 3% DM separated optic nerve proteins into pI fractions 3–5, 5–6.5, 6.5–8, and 8–11. After IEF, 2D PAGE demonstrated distinctly different patterns for each pI fraction. A total of 153 optic nerve proteins were identified by mass spectrometric analysis of select 2D gel spots. For most of these proteins, the sequence calculated pI was in good agreement with the pH range of the trapping chamber from which it was found. Conclusions: Optic nerve contains significant lipid–rich myelin membranes and constitutes one of the more difficult tissues from which to extract and analyze soluble protein. Solution state IEF followed by 2D gel analyses has provided a productive approach for fractionating milligram amounts of optic nerve protein. The proteins identified in this study represent the most extensive catalogue to–date of human optic nerve proteins.

Keywords: proteomics • astroglia: optic nerve head • lamina cribrosa