Coupling Capillary Zone Electrophoresis to a Q Exactive HF Mass Spectrometer for Top-down Proteomics: 580 Proteoform Identifications from Yeast

We used reversed-phase
liquid chromatography to separate the yeast
proteome into 23 fractions. These fractions were then analyzed using
capillary zone electrophoresis (CZE) coupled to a Q-Exactive HF mass
spectrometer using an electrokinetically pumped sheath flow interface.
The parameters of the mass spectrometer were first optimized for top-down
proteomics using a mixture of seven model proteins; we observed that
intact protein mode with a trapping pressure of 0.2 and normalized
collision energy of 20% produced the highest intact protein signals
and most protein identifications. Then, we applied the optimized parameters
for analysis of the fractionated yeast proteome. From this, 580 proteoforms
and 180 protein groups were identified via database searching of the
MS/MS spectra. This number of proteoform identifications is two times
larger than that of previous CZE-MS/MS studies. An additional 3,243
protein species were detected based on the parent ion spectra. Post-translational
modifications including N-terminal acetylation, signal peptide removal,
and oxidation were identified.