Characterization of Protein Variants and Post-Translational Modifications: ESI-MSn Analyses of Intact Proteins Eluted from

We have developed a strategy to characterize protein isoforms, resulting from single-point mutations and post-translational modifications. This strategy is based on polyacrylamide gel electrophoresis separation of protein isoforms, mass spectrometry (MS) and MSn analyses of intact proteins, and tandem MS analyses of proteolytic peptides. We extracted protein isoforms from polyacryl-amide gels by passive elution using SDS, followed by nanoscale hydrophilic phase chromatography for SDS re-moval. We performed electrospray ionization MS analyses of the intact proteins to determine their molecular mass, allowing us to draw hypotheses on the nature of the mod-ification. In the case of labile post-translational modifica-tions, like phosphorylations and glycosylations, we con-ducted electrospray ionization MSn analyses of the intac