title: Benchmarking multiple fragmentation methods on an orbitrap fusion for top-down phospho-proteoform characterization.
abstract: Top-down analysis of intact proteins by mass spectrometry provides an ideal platform for comprehensive proteoform characterization, in particular, for the identification and localization of post-translational modifications (PTM) co-occurring on a protein. One of the main bottlenecks in top-down proteomics is insufficient protein sequence coverage caused by incomplete protein fragmentation. Based on previous work on peptides, increasing sequence coverage and PTM localization by combining sequential ETD and HCD fragmentation in a single fragmentation event, we hypothesized that protein sequence coverage and phospho-proteoform characterization could be equally improved by this new dual fragmentation method termed EThcD, recently been made available on the Orbitrap Fusion. Here, we systematically benchmark the performance of several (hybrid) fragmentation methods for intact protein analysis on an Orbitrap Fusion, using as a model system a 17.5 kDa N-terminal fragment of the mitotic regulator Bora. During cell division Bora becomes multiply phosphorylated by a variety of cell cycle kinases, including Aurora A and Plk1, albeit at distinctive sites. Here, we monitor the phosphorylation of Bora by Aurora A and Plk1, analyzing the generated distinctive phospho-proteoforms by top-down fragmentation. We show that EThcD and ETciD on a Fusion are feasible and capable of providing richer fragmentation spectra compared to HCD or ETD alone, increasing protein sequence coverage, and thereby facilitating phosphosite localization and the determination of kinase specific phosphorylation sites in these phospho-proteoforms. Data are available via ProteomeXchange with identifier PXD001845.
authors: †Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands.|‡Netherlands Proteomics Center, Padualaan 8, 3584CH Utrecht, The Netherlands.
authors: Andrea M
authors: AM
authors: Brunner
authors: †Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands.|‡Netherlands Proteomics Center, Padualaan 8, 3584CH Utrecht, The Netherlands.
authors: Philip
authors: P
authors: Lössl
authors: †Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands.|‡Netherlands Proteomics Center, Padualaan 8, 3584CH Utrecht, The Netherlands.
authors: Fan
authors: F
authors: Liu
authors: §Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, California 95134, United States.
authors: Romain
authors: R
authors: Huguet
authors: §Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, California 95134, United States.
authors: Christopher
authors: C
authors: Mullen
authors: ∥Forschungsgruppe Zelluläre Strukturbiologie, Max-Planck-Institut für Biochemie, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors: Masami
authors: M
authors: Yamashita
authors: §Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, California 95134, United States.
authors: Vlad
authors: V
authors: Zabrouskov
authors: †Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands.|⊥Thermo Fisher Scientific (Bremen) GmbH, Hanna-Kunath-Strasse 11, 28199 Bremen, Germany.
authors: Alexander
authors: A
authors: Makarov
authors: †Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands.|‡Netherlands Proteomics Center, Padualaan 8, 3584CH Utrecht, The Netherlands.
authors: A F Maarten
authors: AF
authors: Altelaar
authors: †Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands.|‡Netherlands Proteomics Center, Padualaan 8, 3584CH Utrecht, The Netherlands.
authors: Albert J R
authors: AJ
authors: Heck
mesh_terms: D064107:Aurora Kinase B / Q000032:analysis* / Q000378:metabolism; D013058:Mass Spectrometry; D010766:Phosphorylation; D040901:Proteomics
chemical_list: D064107:Aurora Kinase B