Capillary Zone Electrophoresis-Electron-Capture Collision-Induced Dissociation on a Quadrupole Time-of-Flight Mass Spectrometer for Top-Down Characterization of Intact Proteins

Mass spectrometry
(MS)-based denaturing top-down proteomics (dTDP)
requires high-capacity separation and extensive gas-phase fragmentation
of proteoforms. Herein, we coupled capillary zone electrophoresis
(CZE) to electron-capture collision-induced dissociation (ECciD) on
an Agilent 6545 XT quadrupole time-of-flight (Q-TOF) mass spectrometer
for dTDP for the first time. During ECciD, the protein ions were first
fragmented using ECD, followed by further activation and fragmentation
by applying a CID potential. In this pilot study, we optimized the
CZE-ECciD method for small proteins (lower than 20 kDa) regarding
the charge state of protein parent ions for fragmentation and the
CID potential applied to maximize the protein backbone cleavage coverage
and the number of sequence-informative fragment ions. The CZE-ECciD
Q-TOF platform provided extensive backbone cleavage coverage for three
standard proteins lower than 20 kDa from only single charge states
in a single CZE-MS/MS run in the targeted MS/MS mode, including ubiquitin
(97%, +7, 8.6 kDa), superoxide dismutase (SOD, 87%, +17, 16 kDa),
and myoglobin (90%, +16, 17 kDa). The CZE-ECciD method produced comparable
cleavage coverage of small proteins (i.e., myoglobin) with direct-infusion
MS studies using electron transfer dissociation (ETD), activated ion-ETD,
and combinations of ETD and collision-based fragmentation on high-end
orbitrap mass spectrometers. The results render CZE-ECciD a new tool
for dTDP to enhance both separation and gas-phase fragmentation of
proteoforms.