10.1021/acs.jproteome.7b00043.s004
Timothy
P. Cleland
Timothy
P.
Cleland
Caroline J. DeHart
Caroline J.
DeHart
Ryan T. Fellers
Ryan T.
Fellers
Alexandra J. VanNispen
Alexandra J.
VanNispen
Joseph B. Greer
Joseph B.
Greer
Richard D. LeDuc
Richard D.
LeDuc
W. Ryan Parker
W. Ryan
Parker
Paul M. Thomas
Paul M.
Thomas
Neil L. Kelleher
Neil L.
Kelleher
Jennifer S. Brodbelt
Jennifer S.
Brodbelt
High-Throughput
Analysis of Intact Human Proteins
Using UVPD and HCD on an Orbitrap Mass Spectrometer
American Chemical Society
2017
elucidate proteoform variation
UVPD
PTM
high-throughput top-down analysis
Intact Human Proteins
Orbitrap Mass Spectrometer
HCD data sets
HeLa whole-cell lysate
protein
sequence coverage
energy collisional dissociation
2017-04-17 00:00:00
Dataset
https://acs.figshare.com/articles/dataset/High-Throughput_Analysis_of_Intact_Human_Proteins_Using_UVPD_and_HCD_on_an_Orbitrap_Mass_Spectrometer/4888649
The analysis of intact proteins (top-down
strategy) by mass spectrometry
has great potential to elucidate proteoform variation, including patterns
of post-translational modifications (PTMs), which may not be discernible
by analysis of peptides alone (bottom-up approach). To maximize sequence
coverage and localization of PTMs, various fragmentation modes have
been developed to produce fragment ions from deep within intact proteins.
Ultraviolet photodissociation (UVPD) has recently been shown to produce
high sequence coverage and PTM retention on a variety of proteins,
with increasing evidence of efficacy on a chromatographic time scale.
However, utilization of UVPD for high-throughput top-down analysis
to date has been limited by bioinformatics. Here we detected 153 proteins
and 489 proteoforms using UVPD and 271 proteins and 982 proteoforms
using higher energy collisional dissociation (HCD) in a comparative
analysis of HeLa whole-cell lysate by qualitative top-down proteomics.
Of the total detected proteoforms, 286 overlapped between the UVPD
and HCD data sets, with 68% of proteoforms having <i>C</i> scores greater than 40 for UVPD and 63% for HCD. The average sequence
coverage (28 ± 20% for UVPD versus 17 ± 8% for HCD, <i>p</i> < 0.0001) was found to be higher for UVPD than HCD
and with a trend toward improvement in <i>q</i> value for
the UVPD data set. This study demonstrates the complementarity of
UVPD and HCD for more extensive protein profiling and proteoform characterization.