title: Implementation of Ultraviolet Photodissociation on a Benchtop Q Exactive Mass Spectrometer and Its Application to Phosphoproteomics.
abstract: Proteomics applications performed on the popular benchtop Q Exactive Orbitrap mass spectrometer have so far relied exclusively on higher collision-energy dissociation (HCD) fragmentation for peptide sequencing. While this fragmentation technique is applicable to a wide range of biological questions, it also has limitations, and all questions cannot be addressed equally well. Here, we demonstrate that the fragmentation capabilities of the Q Exactive mass spectrometer can be extended with ultraviolet photodissociation (UVPD) fragmentation, complete with synchronization triggering to make it compatible with liquid chromatography (LC)/tandem mass spectrometry (MS/MS) workflows. We show that UVPD not only is directly compatible with LC/MS workflows but also, when combined with these workflows, can result in higher database scores and increased identification rates for complex samples as compared to HCD methods. UVPD as a fragmentation technique offers prompt, high-energy fragmentation, which can potentially lead to improved analyses of labile post-translational modifications. Techniques like HCD result in substantial amounts of modification losses, competing with fragmentation pathways that provide information-rich ion fragments. We investigate here the utility of UVPD for identification of phosphorylated peptides and find that UVPD fragmentation reduces the extent of labile modification loss by up to ∼60%. Collectively, when integrated into a complete workflow on the Q Exactive Orbitrap, UVPD provides distinct advantages to the analysis of post-translational modifications and is a powerful and complementary addition to the proteomic toolbox.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University , 3584 Utrecht, The Netherlands.|Netherlands Proteomics Center , 3584 Utrecht, The Netherlands.
authors.forename: Kyle L
authors.initials: KL
authors.lastname: Fort
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University , 3584 Utrecht, The Netherlands.|Netherlands Proteomics Center , 3584 Utrecht, The Netherlands.
authors.forename: Andrey
authors.initials: A
authors.lastname: Dyachenko
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University , 3584 Utrecht, The Netherlands.|Netherlands Proteomics Center , 3584 Utrecht, The Netherlands.
authors.forename: Clement M
authors.initials: CM
authors.lastname: Potel
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University , 3584 Utrecht, The Netherlands.|Netherlands Proteomics Center , 3584 Utrecht, The Netherlands.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Corradini
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University , 3584 Utrecht, The Netherlands.|Netherlands Proteomics Center , 3584 Utrecht, The Netherlands.
authors.forename: Fabio
authors.initials: F
authors.lastname: Marino
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University , 3584 Utrecht, The Netherlands.|Netherlands Proteomics Center , 3584 Utrecht, The Netherlands.
authors.forename: Arjan
authors.initials: A
authors.lastname: Barendregt
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University , 3584 Utrecht, The Netherlands.|Thermo Fisher Scientific (Bremen) , 28199 Bremen, Germany.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Makarov
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University , 3584 Utrecht, The Netherlands.|Netherlands Proteomics Center , 3584 Utrecht, The Netherlands.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Scheltema
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University , 3584 Utrecht, The Netherlands.|Netherlands Proteomics Center , 3584 Utrecht, The Netherlands.
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D002853:Chromatography, Liquid / Q000295:instrumentation; D010750:Phosphoproteins / Q000032:analysis* / Q000528:radiation effects*; D010782:Photolysis / Q000528:radiation effects*; D040901:Proteomics / Q000295:instrumentation*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation*; D014466:Ultraviolet Rays
chemical_list: D010750:Phosphoproteins