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Göteborgs universitets publikationer

Validation of regulated protein phosphorylation events in yeast by quantitative mass spectrometry analysis of purified proteins

Författare och institution:
W. Reiter (-); D. Anrather (-); I. Dohnal (-); P. Pichler (-); J. Veis (-); Morten Grøtli (Institutionen för kemi och molekylärbiologi); F. Posas (-); G. Ammerer (-)
Publicerad i:
Proteomics, 12 ( 19-20 ) s. 3030-3043
Artikel, refereegranskad vetenskaplig
Sammanfattning (abstract):
Global phosphoproteomic studies based on MS have generated qualitative and quantitative data describing protein phosphorylation events in various biological systems. Since high-throughput data for protein modifications are inherently incomplete, we developed a strategy to extend and validate such primary datasets. We selected interesting protein candidates from a global screen in yeast and employed a modified histidine biotin tag that allows tandem affinity purifications of our targets under denaturing conditions. Products in question can be digested directly from affinity resins and phosphopeptides can be further enriched via TiO2 before MS analysis. Our robust protocol can be amended for SILAC as well as iTRAQ quantifications or label-free approaches based on selective reaction monitoring, allowing completion of the phosphorylation pattern in a first step, followed by a detailed analysis of the phosphorylation kinetics. We exemplify the value of such a strategy by an in-depth analysis of Pan1, a highly phosphorylated factor involved in early steps of endocytosis. The study of Pan1 under osmotic stress conditions in different mutant backgrounds allowed us to differentiate between mitogen-activated protein kinase Hog1 driven and Hog1 independent stress responses.
Ämne (baseras på Högskoleverkets indelning av forskningsämnen):
Medicinska grundvetenskaper ->
Cell- och molekylärbiologi
Cell biology, Endocytosis, Phosphorylation, Quantitative proteomics, Signal transduction, serine/threonine kinase prk1p, saccharomyces-cerevisiae, gene-expression, phosphoproteome analysis, global analysis, arp2/3, complex, actin, endocytosis, stress, pan1p
Postens nummer:
Posten skapad:
2012-11-23 12:22

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