Top-Down Proteomics by Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Large-Scale Characterization of Proteoforms in Complex Samples

Abstract

Capillary zone electrophoresis (CZE) is a fundamentally simple and highly efficient separation technique based on differences in electrophoretic mobilities of analytes. CZE-mass spectrometry (MS) has become an important analytical tool in top-down proteomics which aims to delineate proteoforms in cells comprehensively, because of the improvement of capillary coatings, sample stacking methods, and CE-MS interfaces. Here, we present a CZE-MS/MS-based top-down proteomics procedure for the characterization of a standard protein mixture and an Escherichia coli (E. coli) cell lysate using linear polyacrylamide-coated capillaries, a dynamic pH junction sample stacking method, a commercialized electro-kinetically pumped sheath flow CE-MS interface and an Orbitrap mass spectrometer. CZE-MS/MS can identify hundreds of proteoforms routinely from the E. coli sample with a 1% proteoform-level false discovery rate (FDR).