In-line separation by capillary electrophoresis prior to analysis by top-down mass spectrometry enables sensitive characterization of protein complexes

J Proteome Res. 2014 Dec 5;13(12):6078-86. doi: 10.1021/pr500971h. Epub 2014 Nov 21.

Abstract

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.

Keywords: capillary electrophoresis; phosphorylation site mapping; phosphorylation stoichiometry; post-translational modification; protein complexes; top-down mass spectrometry.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Binding Sites
  • Cell Cycle Proteins / analysis*
  • Cell Cycle Proteins / metabolism
  • Electrophoresis, Capillary / methods*
  • Microtubule-Associated Proteins / analysis*
  • Microtubule-Associated Proteins / metabolism
  • Molecular Weight
  • Phosphorylation
  • Protein Subunits / analysis
  • Protein Subunits / metabolism
  • Protein-Serine-Threonine Kinases / metabolism
  • Proteomics / methods*
  • Reproducibility of Results
  • Saccharomyces cerevisiae Proteins / analysis*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Spectrometry, Mass, Electrospray Ionization / methods*

Substances

  • Cell Cycle Proteins
  • DAM1 protein, S cerevisiae
  • Microtubule-Associated Proteins
  • Protein Subunits
  • Saccharomyces cerevisiae Proteins
  • Protein-Serine-Threonine Kinases
  • MPS1 protein, S cerevisiae