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

Academic Article
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Overview

authors

  • Han, X.
  • Wang, Y.
  • Aslanian, A.
  • Fonslow, B.
  • Graczyk, B.
  • Davis, T. N.
  • Yates III, John

publication date

  • 2014

journal

  • Journal of Proteome Research  Journal

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.

subject areas

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

keywords

  • capillary electrophoresis
  • phosphorylation site mapping
  • phosphorylation stoichiometry
  • post-translational modification
  • protein complexes
  • top-down mass spectrometry
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Identity

PubMed Central ID

  • PMC4262260

International Standard Serial Number (ISSN)

  • 1535-3893

Digital Object Identifier (DOI)

  • 10.1021/pr500971h

PubMed ID

  • 25382489
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Additional Document Info

start page

  • 6078

end page

  • 6086

volume

  • 13

issue

  • 12

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