Figure 1
From: In-depth mass spectrometric mapping of the human vitreous proteome
Experimental work flow for vitreous humor proteome analysis. The grey coloured boxes depict the work flow of the standard procedure for proteomic analysis of vitreous humor. After precipitation vitreous proteins were separated by 1D SDS gel electrophoresis. Following Coomassie staining lanes were cut into 26 slices of the same size. Proteins in each individual slice were subjected to in-gel digestion with trypsin. MS/MS data obtained by nanoLC-ESI-MS/MS analysis were searched with the MASCOT search engine against SwissProt database. In order to test alternative experimental strategies different variants of the standard procedure were applied. Variant 1: Liquid phase IEF was used as additional prefractionation step before SDS-gel electrophoresis. Variant 2: Samples obtained from liquid phase IEF were directly applied for nanoLC-ESI-MS/MS analysis. Variant 3: Vitreous proteins obtained after TCA/acetone precipitation was directly subjected to nanoLC-ESI-MS/MS analysis without any prefractionation.