In Depth Analysis of Retinal Proteins using a Combinatory HPLC based Mass Spectrometric Platform

Abstract

Purpose: The retina proteome with special focus on retinal ganglion cell proteins is of important interest regarding clinical research on neurodegenerative diseases like glaucoma. To intensively characterize retinal proteins we developed protein extraction and fractionation techniques coupled to LC MALDI and ESI methods to increase the sensitivity towards retinal sample species. Detailed reference maps should be generated by use of the developed workflow providing new insights to the understanding of the retina proteome.

Methods: Retinal sample species were used as study material. Different extraction techniques were developed encircling non-ionic detergent extraction, stepwise differential extraction and methanol-chloroform extraction for sample fractionation. Extract analysis includes gel-based RP-RP-2D-capillary-LC MALDI TOF/TOF and RP-capillary LC ESI-LTQ Orbitrap mass spectrometry. The medium molecular weight range was directly investigated by top down microbore-LC MALDI TOF MS. Results were used to generate reference protein maps of so far identified retinal proteins.

Results: By use of non-ionic detergent extraction more than 1000 proteins per lysate could be identified with high reproducibility by the LC ESI approach in a single sample lysate.The gel-based MALDI approaches detected more than 300 proteins. More than 856 medium molecular weight components per sample were detected by top down MALDI analysis. Furthermore ESI and MALDI methods in combination could distinctly increase the output emphasizing the complementary power of MALDI and ESI techniques. By use of stepwise differential extraction and chloroform-methanol extraction a distinct and reproducible fractionation of proteins could be achieved. Moreover post-translational modification sites of retinal proteins have been determined by mass spectrometric analysis.

Conclusions: The use of the developed platform allowed us to get a deep view to the retina proteome providing detailed reference maps. Accordingly we could demonstrate the promising use of fractionation techniques coupled to HPLC based mass spectrometric approaches for retina related sample species. We think that the provided reference maps as well as the developed workflow will contribute to clinical retina research.

Keywords: 663 proteomics • 688 retina