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LC-MS Quantification of Site-Specific Phosphorylation Degree by Stable-Isotope Dimethyl Labeling Coupled with Phosphatase Dephosphorylation.
Chen, Sin-Hong; Lin, Ya-Chi; Shih, Ming-Kuei; Wang, Li-Fei; Liu, Shyh-Shyan; Hsu, Jue-Liang.
Afiliación
  • Chen SH; Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.
  • Lin YC; Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.
  • Shih MK; Graduate Institute of Food Culture and Innovation, National Kaohsiung University of Hospitality and Tourism, Kaohsiung 81271, Taiwan.
  • Wang LF; Hospitality and Tourism Research Center, National Kaohsiung University of Hospitality and Tourism, Kaohsiung 81271, Taiwan.
  • Liu SS; Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.
  • Hsu JL; Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.
Molecules ; 25(22)2020 Nov 14.
Article en En | MEDLINE | ID: mdl-33202651
Protein phosphorylation is a crucial post-translational modification that plays an important role in the regulation of cellular signaling processes. Site-specific quantitation of phosphorylation levels can help decipher the physiological functions of phosphorylation modifications under diverse physiological statuses. However, quantitative analysis of protein phosphorylation degrees is still a challenging task due to its dynamic nature and the lack of an internal standard simultaneously available for the samples differently prepared for various phosphorylation extents. In this study, stable-isotope dimethyl labeling coupled with phosphatase dephosphorylation (DM + deP) was tried to determine the site-specific degrees of phosphorylation in proteins. Firstly, quantitation accuracy of the (DM + deP) approach was confirmed using synthetic peptides of various simulated phosphorylation degrees. Afterwards, it was applied to evaluate the phosphorylation stoichiometry of milk caseins. The phosphorylation degree of Ser130 on α-S1-casein was also validated by absolute quantification with the corresponding synthetic phosphorylated and nonphosphorylated peptides under a selected reaction monitoring (SRM) mode. Moreover, this (DM + deP) method was used to detect the phosphorylation degree change of Ser82 on the Hsp27 protein of HepG2 cells caused by tert-butyl hydroperoxide (t-BHP) treatment. The results showed that the absolute phosphorylation degree obtained from the (DM + deP) approach was comparable with the relative quantitation resulting from stable-isotope dimethyl labeling coupled with TiO2 enrichment. This study suggested that the (DM + deP) approach is promising for absolute quantification of site-specific degrees of phosphorylation in proteins, and it may provide more convincing information than the relative quantification method.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Monoéster Fosfórico Hidrolasas / Espectrometría de Masas en Tándem / Marcaje Isotópico Límite: Humans Idioma: En Revista: Molecules Asunto de la revista: BIOLOGIA Año: 2020 Tipo del documento: Article País de afiliación: Taiwán Pais de publicación: Suiza
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Monoéster Fosfórico Hidrolasas / Espectrometría de Masas en Tándem / Marcaje Isotópico Límite: Humans Idioma: En Revista: Molecules Asunto de la revista: BIOLOGIA Año: 2020 Tipo del documento: Article País de afiliación: Taiwán Pais de publicación: Suiza