Identification of phosphorylation sites on phosducin-like protein by QTOF mass spectrometry.
J Biomol Tech
; 15(4): 257-64, 2004 Dec.
Article
en En
| MEDLINE
| ID: mdl-15585822
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.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Proteínas Portadoras
/
Técnicas de Química Analítica
/
Proteínas del Tejido Nervioso
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
En
Revista:
J Biomol Tech
Asunto de la revista:
BIOTECNOLOGIA
Año:
2004
Tipo del documento:
Article
País de afiliación:
Estados Unidos