title: Identification of phosphorylation sites on phosducin-like protein by QTOF mass spectrometry.
abstract: Post-translational modifications are used by cells to control the functions of proteins. Phosducin-like protein (PhLP) is a regulator of G-protein signaling that is post-translationally modified via phosphorylation. Phosphorylation of PhLP initiates its degradation by the 26S proteasome in serum-stimulated cells. In this report, we show that PhLP is phosphorylated in serum-stimulated Chinese hamster ovary (CHO) cells. Through the use of tandem mass spectrometry (MS/MS), the specific amino acids phosphorylated can be identified. A PhLP-myc-His construct was purified and phosphorylated by serum-stimulated CHO extract. The resulting protein was digested with trypsin and the peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Automated collison-induced dissociation data acquisition was compared with LC-MS/MS of manually chosen parents. In general, LC-MS/MS is superior for parent ions chosen manually, with the notable exception that automated fragmentation employs dynamic collision energy, which can result in higher quality collison-induced dissociation. Using the LC-MS/MS methods, four phosphorylation sites on PhLP were positively identified.
authors.affiliation: Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Carter
authors.forename: Katie
authors.initials: K
authors.lastname: Southwick
authors.forename: Georgi
authors.initials: G
authors.lastname: Lukov
authors.forename: Barry M
authors.initials: BM
authors.lastname: Willardson
authors.forename: Craig D
authors.initials: CD
authors.lastname: Thulin
mesh_terms: D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry*; D002623:Chemistry Techniques, Analytical; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010766:Phosphorylation
chemical_list: D002352:Carrier Proteins; D009419:Nerve Tissue Proteins