Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry

Successful high-throughput
characterization of intact proteins
from complex biological samples by mass spectrometry requires instrumentation
capable of high mass resolving power, mass accuracy, sensitivity,
and spectral acquisition rate. These limitations often necessitate
the performance of hundreds of LC–MS/MS experiments to obtain
reasonable coverage of the targeted proteome, which is still typically
limited to molecular weights below 30 kDa. The National High Magnetic
Field Laboratory (NHMFL) recently installed a 21 T FT-ICR mass spectrometer,
which is part of the NHMFL FT-ICR User Facility and available to all
qualified users. Here we demonstrate top-down LC-21 T FT-ICR MS/MS
of intact proteins derived from human colorectal cancer cell lysate.
We identified a combined total of 684 unique protein entries observed
as 3238 unique proteoforms at a 1% false discovery rate, based on
rapid, data-dependent acquisition of collision-induced and electron-transfer
dissociation tandem mass spectra from just 40 LC–MS/MS experiments.
Our identifications included 372 proteoforms with molecular weights
over 30 kDa detected at isotopic resolution, which substantially extends
the accessible mass range for high-throughput top-down LC–MS/MS.