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

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

Author information

1
Department of Chemical Physiology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

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

PMID:
25382489
PMCID:
PMC4262260
DOI:
10.1021/pr500971h
[Indexed for MEDLINE]
Free PMC Article

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