title: 193 nm Ultraviolet Photodissociation Mass Spectrometry for Phosphopeptide Characterization in the Positive and Negative Ion Modes.
abstract: Advances in liquid chromatography tandem mass spectrometry (LC-MS/MS) have permitted phosphoproteomic analysis on a grand scale, but ongoing challenges specifically associated with confident phosphate localization continue to motivate the development of new fragmentation techniques. In the present study, ultraviolet photodissociation (UVPD) at 193 nm is evaluated for the characterization of phosphopeptides in both positive and negative ion modes. Compared to the more standard higher energy collisional dissociation (HCD), UVPD provided more extensive fragmentation with improved phosphate retention on product ions. Negative mode UVPD showed particular merit for detecting and sequencing highly acidic phosphopeptides from alpha and beta casein, but was not as robust for larger scale analysis because of lower ionization efficiencies in the negative mode. HeLa and HCC70 cell lysates were analyzed by both UVPD and HCD. While HCD identified more phosphopeptides and proteins compared to UVPD, the unique matches from UVPD analysis could be combined with the HCD data set to improve the overall depth of coverage compared to either method alone.
authors.affiliation: Department of Chemistry, and ‡Division of Chemical Biology and Medicinal Chemistry College of Pharmacy, The University of Texas Austin, Texas 78712, United States.
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Robinson
authors.affiliation: Department of Chemistry, and ‡Division of Chemical Biology and Medicinal Chemistry College of Pharmacy, The University of Texas Austin, Texas 78712, United States.
authors.forename: Juliana M
authors.initials: JM
authors.lastname: Taliaferro
authors.affiliation: Department of Chemistry, and ‡Division of Chemical Biology and Medicinal Chemistry College of Pharmacy, The University of Texas Austin, Texas 78712, United States.
authors.forename: Kevin N
authors.initials: KN
authors.lastname: Dalby
authors.affiliation: Department of Chemistry, and ‡Division of Chemical Biology and Medicinal Chemistry College of Pharmacy, The University of Texas Austin, Texas 78712, United States.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Brodbelt
mesh_terms: D006367:HeLa Cells; D006801:Humans; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods; D010446:Peptide Fragments; D010748:Phosphopeptides / Q000032:analysis*; D010782:Photolysis / Q000528:radiation effects*; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods; D014466:Ultraviolet Rays
chemical_list: D007477:Ions; D010446:Peptide Fragments; D010748:Phosphopeptides; D011506:Proteins
keywords: higher energy collision induced dissociation; mass spectrometry; phosphoproteomics; ultraviolet photodissociation