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S. Zhou, H. Yang, O. Ou, M. J. Gordon, T. Zhang and J. H. Beattie
Posters: J Proteomics Bioinform
The plasma proteome has a large dynamic range of individual protein concentrations (10 orders of magnitude). Identification of low copy number proteins of interestusing 2-DE is therefore difficult due to the confounding presence of higher abundance proteins and adversely affects biomarker discovery. Several columns have been developed to deplete the higher abundance proteins. For example, the Seppro Column has frequently been used in the published literature. It can remove the top 7 highly abundant proteins in mouse plasma, and be repeatedly used up to 80 times, according to the manufacturer?s documentation. However, there are noindependent data on the efficiency of protein extraction after repeated use of the column. The aim of the study is to 1) optimize the concentration methods of depleted protein sample; 2) to test the efficiency of the column after 50 depletion cycles for abundant protein removal.The proteins of the depleted fraction, the non-specifically bound protein, and the specifically-bound protein were measured after Seppro spin column treatment. Protein recovery at each step was: (1) depleted protein 27.7%; (2) non-specifically-bound protein 2.4%; (3) bound protein 25.2%. Protein was further concentrated byusing Vivaspin4 5kDa MWCO membranes, trichroroacetic acid (TCA) precipitation,and a RedayPrep 2-D Cleanup Kit.The protein recovery rate was 60%, 67%, and 76%,respectively.200μg of depleted or bound protein from Vivaspin4 5kDa MWCO, TCA precipitation was loaded onto pH 3-10 IPG strips (18cm) and 8-16% SDS gels in the second dimension. The gels were stained with Coomassie blue and analysed by Progenesis SameSpots software. The number of protein spotswas 600 for crude plasma precipitated with TCA, 604 for depleted protein precipitated with TCA, 565 for depleted protein concentrated by vivaspin4 5kDa MWCO, 495 for the bound protein with TCA and 659 spots for the bound protein with vivaspin4 5kDa MWCO concentration. Results also showed that the ratios of depleted protein to total recovered proteindecreased from 70% to 60% after 25 depletions and then gradually decreased to 45% after 50 sample depletions.