pr7b00043_si_004.xlsx (28.81 kB)
High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer
dataset
posted on 2017-04-17, 00:00 authored by Timothy
P. Cleland, Caroline J. DeHart, Ryan T. Fellers, Alexandra J. VanNispen, Joseph B. Greer, Richard D. LeDuc, W. Ryan Parker, Paul M. Thomas, Neil L. Kelleher, Jennifer S. BrodbeltThe 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 C scores greater than 40 for UVPD and 63% for HCD. The average sequence
coverage (28 ± 20% for UVPD versus 17 ± 8% for HCD, p < 0.0001) was found to be higher for UVPD than HCD
and with a trend toward improvement in q value for
the UVPD data set. This study demonstrates the complementarity of
UVPD and HCD for more extensive protein profiling and proteoform characterization.