A novel fractionation method prior to MS-based proteomics analysis using cascade biomimetic affinity chromatography

Abstract

This is the first report that combines cascade biomimetic affinity fractionation with MS-based proteomics analysis. Our lab has constructed an affinity ligand library composed of thousands of ligands with different protein-binding properties. Structural differences between these ligands result in different non-bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first screened out three affinity ligands with large difference in protein-binding properties. Next, cascade combination of these ligands was applied to fractionate tissue sample into simple subgroups prior to trypsin digestion and LC–MS/MS analysis. In this study, 391 non-redundant protein groups were identified in unfractionated rat liver cytosol, 499 protein groups were identified in 2 fractions of the first affinity fractionation, 616 in 4 fractions of the second fractionation, and 738 in 8 fractions of the third fractionation (an 88.74% increase). Ultimately, a total of 859 unique protein groups were identified in all cascade fractions (a 119.6% increase compared with unfractionated sample). The proteins detected in each fraction were bioinformatically categorized according to their physicochemical characteristics (relative molecular mass, p I, GRAVY value and TM helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes.