Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry for Top-Down Characterization of Pyrococcus furiosus Proteins on a Proteome Scale

Intact protein analysis via top-down mass spectrometry (MS) provides the unique capability of fully characterizing protein isoforms and combinatorial post-translational modifications (PTMs) compared to the bottom-up MS approach. Front-end protein separation poses a challenge for analyzing complex mi...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2014-11, Vol.86 (22), p.11006-11012
Main Authors: Han, Xuemei, Wang, Yueju, Aslanian, Aaron, Bern, Marshall, Lavallée-Adam, Mathieu, Yates, John R
Format: Article
Language:English
Subjects:
Acetylation
Analytical chemistry
Archaeal Proteins - analysis
Capillarity
Combinatorial analysis
Consumption
Electrophoresis, Capillary
Glycosylation
Ionization
Letter
Mass spectrometers
Mass spectrometry
Oxidation
Proteins
Proteome - analysis
Proteomics
Pyrococcus furiosus
Pyrococcus furiosus - chemistry
Tandem Mass Spectrometry
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Summary:Intact protein analysis via top-down mass spectrometry (MS) provides the unique capability of fully characterizing protein isoforms and combinatorial post-translational modifications (PTMs) compared to the bottom-up MS approach. Front-end protein separation poses a challenge for analyzing complex mixtures of intact proteins on a proteomic scale. Here we applied capillary electrophoresis (CE) through a sheathless capillary electrophoresis-electrospray ionization (CESI) interface coupled to an Orbitrap Elite mass spectrometer to profile the proteome from Pyrococcus furiosus. CESI-top-down MS analysis of Pyrococcus furiosus cell lysate identified 134 proteins and 291 proteoforms with a total sample consumption of 270 ng in 120 min of total analysis time. Truncations and various PTMs were detected, including acetylation, disulfide bonds, oxidation, glycosylation, and hypusine. This is the largest scale analysis of intact proteins by CE-top-down MS to date.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac503439n