Quantitation of changes in protein phosphorylation : a simple method based on stable isotope labeling and mass spectrometry

Bonenfant, D. and Schmelzle, T. and Jacinto, E. and Crespo, J. L. and Mini, T. and Hall, M. N. and Jenoe, P.. (2003) Quantitation of changes in protein phosphorylation : a simple method based on stable isotope labeling and mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America, 100 (3). pp. 880-885.

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Official URL: http://edoc.unibas.ch/dok/A5258308

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Reversible protein phosphorylation plays an important role in many cellular processes. However, a simple and reliable method to measure changes in the extent of phosphorylation is lacking. Here, we present a method to quantitate the changes in phosphorylation occurring in a protein in response to a stimulus. The method consists of three steps: (i) enzymatic digestion in H(2)16O or isotopically enriched H(2)18O to label individual pools of differentially phosphorylated proteins; (ii) affinity selection of phosphopeptides from the combined digests by immobilized metal-affinity chromatography; and (iii) dephosphorylation with alkaline phosphatase to allow for quantitation of changes of phosphorylation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We applied this strategy to the analysis of the yeast nitrogen permease reactivator protein kinase involved in the target of rapamycin signaling pathway. Alteration in the extent of phosphorylation at Ser-353 and Ser-357 could be easily assessed and quantitated both in wild-type yeast cells treated with rapamycin and in cells lacking the SIT4 phosphatase responsible for dephosphorylating nitrogen permease reactivator protein. The method described here is simple and allows quantitation of relative changes in the level of phosphorylation in signaling proteins, thus yielding information critical for understanding the regulation of complex protein phosphorylation cascades.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Hall)
05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Mass Spectrometry (Jenö)
UniBasel Contributors:Hall, Michael N. and Jenö, Paul
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:National Academy of Sciences
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:09 Nov 2017 07:15
Deposited On:22 Mar 2012 13:20

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