Prefractionation of Intact Proteins by Reversed-Phase and Anion-Exchange Chromatography for the Differential Proteomic Analysis of Saccharomyces cerevisiae

The need for multidimensional separations for bottom-up proteomic analyses has been well demonstrated by many over the past decade. The vast majority of reported approaches has focused primarily on improving the separation once the sample has already been digested. The work presented in this study shows an improvement in multidimensional approaches by prefractionation of intact proteins prior to digestion and separation of the peptides. Two modes of intact protein separation were compared, anion-exchange and reversed-phase, to assess the utility of each mode for the purpose of fractionation. Each of the samples was then enzymatically digested and analyzed by RP-UPLC-MSE. To assess the validity of each approach, baker’s yeast (Saccharomyces cerevisiae) was grown on two different carbon sources, glycerol and dextrose. More proteins were identified by the reversed-phase prefractionation approach (546) than were found by the anion-exchange method (262). As a result, there was much greater coverage of the metabolic pathways of interest for the reversed-phase method than for the anion-exchange method.