Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer

Over
the past decade, developments in high resolution mass spectrometry
have enabled the high throughput analysis of intact proteins from
complex proteomes, leading to the identification of thousands of proteoforms.
Several previous reports on top-down proteomics (TDP) relied on hybrid
ion trap–Fourier transform mass spectrometers combined with
data-dependent acquisition strategies. To further reduce TDP to practice,
we use a quadrupole-Orbitrap instrument coupled with software for
proteoform-dependent data acquisition to identify and characterize
nearly 2000 proteoforms at a 1% false discovery rate from human fibroblasts.
By combining a 3 m/z isolation window
with short transients to improve specificity and signal-to-noise for
proteoforms >30 kDa, we demonstrate improving proteome coverage
by
capturing 439 proteoforms in the 30–60 kDa range. Three different
data acquisition strategies were compared and resulted in the identification
of many proteoforms not observed in replicate data-dependent experiments.
Notably, the data set is reported with updated metrics and tools including
a new viewer and assignment of permanent proteoform record identifiers
for inclusion of highly characterized proteoforms (i.e., those with
C-scores >40) in a repository curated by the Consortium for Top-Down
Proteomics.