Proteomic Profiling of Intact Proteins Using WAX-RPLC 2-D Separations and FTICR Mass Spectrometry

We investigated the combination of weak anion exchange (WAX) fractionation and on-line reversed-phase liquid chromatography (RPLC) separation using a 12 T FTICR mass spectrometer for the detection
of intact proteins from a Shewanella oneidensis MR-1 cell lysate. This work aimed at optimizing intact
protein detection for profiling proteins at a level that incorporates their modification state. A total of
715 intact proteins were detected, and the combined results from the WAX fractions and the
unfractionated cell lysate were aligned using LC-MS features to facilitate protein abundance measurements. Protein identifications and post-translational modifications were assigned for ∼10% of the
detected proteins by comparing intact protein mass measurements to proteins identified in peptide
MS/MS analysis of an aliquot of the same fraction. Intact proteins were also detected for S. oneidensis
lysates obtained from cells grown on 13C-, 15N-depleted media under aerobic and sub-oxic conditions.
The strategy can be readily applied for measuring differential protein abundances and provides a
platform for high-throughput selection of biologically relevant targets for further characterization.
Keywords: Comparative proteomics • FTICR MS • intact proteins • post-translational modifications