Identification of phosphorylation sites on phosducin-like protein by QTOF mass spectrometry. |
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Authors: | Michael D Carter Katie Southwick Georgi Lukov Barry M Willardson Craig D Thulin |
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Affiliation: | Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA. |
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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. |
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Keywords: | phosducin-like protein phosphorylation CHO serum-stimulation LC-MS/MS |
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