PEPPI-MS: Polyacrylamide-Gel-Based
Prefractionation
for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry
Version 2 2020-08-06, 21:58
Version 1 2020-07-11, 12:03
Posted on 2020-08-06 - 21:58
Prefractionation
of complex mixtures of proteins derived
from biological samples is indispensable for proteome analysis via
top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis
(PAGE), which enables high-resolution protein separation based on
molecular size, is a widely used technique in biochemical experiments
and has the potential to be useful in sample fractionation for top-down
MS analysis. However, the lack of a means to efficiently recover the
separated proteins in-gel has always been a barrier to its use in
sample prefractionation. In this study, we present a novel experimental
workflow, called Passively Eluting Proteins from Polyacrylamide gels
as Intact species for MS (“PEPPI-MS”), which allows
top-down MS of PAGE-separated proteins. The optimization of Coomassie
brilliant blue staining followed by the passive extraction step in
the PEPPI-MS workflow enabled the efficient recovery of proteins,
separated on commercial precast gels, from a wide range of molecular
weight regions in under 10 min. Two-dimensional separation combining
offline PEPPI-MS with online reversed-phase liquid chromatographic
separation resulted in identification of over 1000 proteoforms recovered
from the target region of the gel (≤50 kDa). Given the widespread
availability and relatively low cost of traditional sodium dodecyl
sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful
prefractionation strategy for top-down proteomics.
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Takemori, Ayako; Butcher, David S.; Harman, Victoria M.; Brownridge, Philip; Shima, Keisuke; Higo, Daisuke; et al. (2020). PEPPI-MS: Polyacrylamide-Gel-Based
Prefractionation
for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry. ACS Publications. Collection. https://doi.org/10.1021/acs.jproteome.0c00303
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AUTHORS (15)
AT
Ayako Takemori
DB
David S. Butcher
VH
Victoria M. Harman
PB
Philip Brownridge
KS
Keisuke Shima
DH
Daisuke Higo
JI
Jun Ishizaki
HH
Hitoshi Hasegawa
JS
Junpei Suzuki
MY
Masakatsu Yamashita
JL
Joseph A. Loo
RL
Rachel R. Ogorzalek Loo
RB
Robert J. Beynon
LA
Lissa C. Anderson
NT
Nobuaki Takemori