title: Phosphoproteomics with Activated Ion Electron Transfer Dissociation.
abstract: The ability to localize phosphosites to specific amino acid residues is crucial to translating phosphoproteomic data into biological meaningful contexts. In a companion manuscript ( Anal. Chem. 2017 , DOI: 10.1021/acs.analchem.7b00213 ), we described a new implementation of activated ion electron transfer dissociation (AI-ETD) on a quadrupole-Orbitrap-linear ion trap hybrid MS system (Orbitrap Fusion Lumos), which greatly improved peptide fragmentation and identification over ETD and other supplemental activation methods. Here we present the performance of AI-ETD for identifying and localizing sites of phosphorylation in both phosphopeptides and intact phosphoproteins. Using 90 min analyses we show that AI-ETD can identify 24,503 localized phosphopeptide spectral matches enriched from mouse brain lysates, which more than triples identifications from standard ETD experiments and outperforms ETcaD and EThcD as well. AI-ETD achieves these gains through improved quality of fragmentation and MS/MS success rates for all precursor charge states, especially for doubly protonated species. We also evaluate the degree to which phosphate neutral loss occurs from phosphopeptide product ions due to the infrared photoactivation of AI-ETD and show that modifying phosphoRS (a phosphosite localization algorithm) to include phosphate neutral losses can significantly improve localization in AI-ETD spectra. Finally, we demonstrate the utility of AI-ETD in localizing phosphosites in α-casein, an ∼23.5 kDa phosphoprotein that showed eight of nine known phosphorylation sites occupied upon intact mass analysis. AI-ETD provided the greatest sequence coverage for all five charge states investigated and was the only fragmentation method to localize all eight phosphosites for each precursor. Overall, this work highlights the analytical value AI-ETD can bring to both bottom-up and top-down phosphoproteomics.
authors.forename: Nicholas M
authors.identifier: 0000-0002-1536-2966
authors.initials: NM
authors.lastname: Riley
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Hebert
authors.affiliation: Institute of Molecular Pathology (IMP) , Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria.|GMI, Gregor Mendel Institute of Molecular Plant Biology , Dr. Bohr Gasse 3, A-1030 Vienna, Austria.|IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences , Dr. Bohr Gasse 3, A-1030 Vienna, Austria.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Dürnberger
authors.affiliation: Institute of Molecular Pathology (IMP) , Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria.
authors.forename: Florian
authors.initials: F
authors.lastname: Stanek
authors.affiliation: Institute of Molecular Pathology (IMP) , Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria.|IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences , Dr. Bohr Gasse 3, A-1030 Vienna, Austria.
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
authors.forename: Michael S
authors.initials: MS
authors.lastname: Westphall
authors.affiliation: Morgridge Institute for Research , Madison, Wisconsin 53715, United States.
authors.forename: Joshua J
authors.identifier: 0000-0002-0004-8253
authors.initials: JJ
authors.lastname: Coon
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D002853:Chromatography, Liquid; D004579:Electron Transport; D007477:Ions / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D010748:Phosphopeptides / Q000737:chemistry* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D007477:Ions; D010748:Phosphopeptides; D010750:Phosphoproteins