Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry
for Top-Down Characterization of <i>Pyrococcus furiosus</i> Proteins on a Proteome Scale
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Abstract
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 <i>Pyrococcus furiosus</i>. CESI-top-down MS analysis
of <i>Pyrococcus furiosus</i> 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