Methodological Analyses and Selective Characterisation of the Neuroretinal Proteome

Abstract

Abstract: : Purpose: In the post genomics era neuroretinal proteomics will be of increasing importance, as this will enable the characterisation of peptide and protein products and the specific post-translational modifications. The identified proteins may be developed as therapies applicable in retinitis pigmentosa, glaucoma and age-related macular degeneration. This study aims to elucidate differential and sequential protein solvation, distinct buffer systems allied with size exclusion and reverse phase HPLC (rpHPLC) and latterly electrospray ionisation quadrupole mass spectrometry (ESI-MS) to characterise neuroretinal proteins. Methods: Porcine retinae (n=20) were dissected and homogenised in a pestle and mortar under liquid nitrogen prior to extraction by a range of protocols. In brief, retinae (0.1 g) were shaken overnight at 4 C in 1 ml of 40 mM Tris, MES, CAPS, MOPS, Hepes, ammonium bicarbonate, 0.5 M acetic acid and acid ethanol. The samples were centrifuged, the supernatant decanted, lyophilised and reconstituted in 500 µl 40 mM sodium acetate and subject to size exclusion HPLC (TOSOHAAS Vydac G4000SW; fractionation range 20 - 10000x10₃ Da; flow rate, 0.5 ml/min) employing a 40 mM sodium acetate containing 0.5 M sodium chloride mobile phase. Proteins with a defined molecular range were subject to reverse phase HPLC employing a range of column chemistries (C4, C8, C16, C18). Absorbancies were measured at 214 nM (peptide bond) and 280 nM (aromatic residues). Results: Each extraction protocol solubilised protein products that were quantified by a protein estimation assay (Pierce BCA Protein Assay; Table 1) and correlated with the predicted cellular proteome profile. Size exclusion chromatographic profiles of each extract resolved a distinct signature. rpHPLC further resolved the protein products which were subject to LC-MS. Conclusion: This study has revealed the distinct and subtle variation in protein recovery utilising chemically distinct extraction protocols. In addition, it has demonstrated that size exclusion and rpHPLC are complementary tools that facilitate the characterisation of the neuroretinal proteome. Table 1  

Keywords: 526 protein purification and characterization • 554 retina • 488 neuropeptides