title: Coupling capillary zone electrophoresis with electron transfer dissociation and activated ion electron transfer dissociation for top-down proteomics.
abstract: Top-down proteomics offers the potential for full protein characterization, but many challenges remain for this approach, including efficient protein separations and effective fragmentation of intact proteins. Capillary zone electrophoresis (CZE) has shown great potential for separation of intact proteins, especially for differentially modified proteoforms of the same gene product. To date, however, CZE has been used only with collision-based fragmentation methods. Here we report the first implementation of electron transfer dissociation (ETD) with online CZE separations for top-down proteomics, analyzing a mixture of four standard proteins and a complex protein mixture from the Mycobacterium marinum bacterial secretome. Using a multipurpose dissociation cell on an Orbitrap Elite system, we demonstrate that CZE is fully compatible with ETD as well as higher energy collisional dissociation (HCD), and that the two complementary fragmentation methods can be used in tandem on the electrophoretic time scale for improved protein characterization. Furthermore, we show that activated ion electron transfer dissociation (AI-ETD), a recently introduced method for enhanced ETD fragmentation, provides useful performance with CZE separations to greatly increase protein characterization. When combined with HCD, AI-ETD improved the protein sequence coverage by more than 200% for proteins from both standard and complex mixtures, highlighting the benefits electron-driven dissociation methods can add to CZE separations.
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 Biomolecular Chemistry, Genome Center of Wisconsin, and Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States.
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Riley
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 Biomolecular Chemistry, Genome Center of Wisconsin, and Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Hebert
authors.affiliation: †Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Yan
authors.affiliation: ‡Department of Biomolecular Chemistry, Genome Center of Wisconsin, and Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Westphall
authors.affiliation: ‡Department of Biomolecular Chemistry, Genome Center of Wisconsin, and Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States.
authors.forename: Matthew J P
authors.initials: MJ
authors.lastname: Rush
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: Matthew M
authors.initials: MM
authors.lastname: Champion
authors.affiliation: §Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Felix
authors.initials: F
authors.lastname: Mba Medie
authors.affiliation: §Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Patricia A DiGiuseppe
authors.initials: PA
authors.lastname: Champion
authors.affiliation: ‡Department of Biomolecular Chemistry, Genome Center of Wisconsin, and Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
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: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004579:Electron Transport; D019075:Electrophoresis, Capillary / Q000379:methods*; D008969:Molecular Sequence Data; D019910:Mycobacterium marinum / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins