title: 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 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Yimeng
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Guijie
authors.initials: G
authors.lastname: Zhu
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods; D005656:Fungal Proteins / Q000032:analysis; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D005656:Fungal Proteins; D020543:Proteome
keywords: RPLC fractionation; capillary zone electrophoresis; electrokinetically pumped nanoelectrospray; post-translational modifications; proteoforms; top-down proteomics; yeast