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

Abstract

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 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. 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 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.

Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms

“…[6] Later, Zhao et al further applied the CZE-MS/MS system for top-down proteomics of Mycobacterium marinum secretome and yeast proteome. [7, 8] Coupling offline RPLC fractionation to CZE-MS/MS identified 580 proteoforms from a yeast lysate. In total, 23 RPLC fractions were analyzed by CZE-MS/MS and up to 180 proteoforms could be identified with single-shot CZE-MS/MS.…”

“…In total, 23 RPLC fractions were analyzed by CZE-MS/MS and up to 180 proteoforms could be identified with single-shot CZE-MS/MS. [8] Li et al developed a CZE-MS system based on the electrokinetically pumped sheath flow interface and applied the system to a complex proteome sample for characterization of large proteins (30–80 kDa), resulting in identification of 30 proteins in the mass range of 30–80 kDa. [9] …”

“…First, the largest sample loading capacity of CZE-MS/MS systems reported in the literature for top-down proteomics is only about 200 nL. [8,10] The low sample loading capacity impedes identification of low abundant proteoforms from complex proteome samples. Second, the reported separation window of CZE-MS/MS systems for top-down proteomics is roughly 30 min.…”

“…Second, the reported separation window of CZE-MS/MS systems for top-down proteomics is roughly 30 min. [8,10] The narrow separation window limits the number of MS/MS spectra acquired during one experiment, which restricts the number of proteoform identifications (IDs) from CZE-MS/MS. Capillary isoelectric focusing (cIEF)-MS is a promising technique for large-scale top-down proteomics due to its large sample loading capacity and high resolution for separation of intact proteins.…”

“…Zhao et al performed top-down proteomics of a yeast lysate using dynamic pH junction based CZE-MS/MS. [8] They used 5 mM ammonium bicarbonate (pH 8) as the sample buffer and 5% (v/v) acetic acid (pH 2.4) as the BGE. 100–240 nL of the sample was injected for CZE-MS/MS analysis.…”