In-Line Separation by Capillary Electrophoresis Prior to Analysis by Top-Down Mass Spectrometry Enables Sensitive Characterization of Protein Complexes

Intact
protein analysis via top-down mass spectrometry (MS) provides
a bird’s eye view over the protein complexes and complex protein
mixtures with the unique capability of characterizing protein variants,
splice isoforms, and combinatorial post-translational modifications
(PTMs). Here we applied capillary electrophoresis (CE) through a sheathless
CE–electrospray ionization interface coupled to an LTQ Velos
Orbitrap Elite mass spectrometer to analyze the Dam1 complex from Saccharomyces cerevisiae. We achieved a 100-fold
increase in sensitivity compared to a reversed-phase liquid chromatography
coupled MS analysis of recombinant Dam1 complex with a total loading
of 2.5 ng (12 amol). N-terminal processing forms of individual subunits
of the Dam1 complex were observed as well as their phosphorylation
stoichiometry upon Mps1p kinase treatment.