Proteomics

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Quantitiative phosphoproteomic analysis of SKS1 signaling network involved in yeast filamentous growth by LC-MS in combination with SILAC


ABSTRACT: Yeast pseudohyphal growth(filamentous growth) is regulated by signaling pathways responsive to nitrogen stress and glucose deprivation, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. To identify the Sks1p signaling network involved in filamentous growth, we applied mass spectrometry-based quantitative phosphoproteomics in combination with SILAC. We investigated differential protein expressions and changes in phosphorylation levels in SKS1 kinase dead mutant yeast cells (SKS1-KD, Lys-4 and Arg-6 labeled) under filamentous growth condition. Twelve SCX fractions of SILAC paired peptides derived from SKS1-KD and wild type yeast cells were subjected to phosphopeptide enrichment by using ZrO2, prior to LC-MSMS by an LTQ-Orbitrap. MaxQunat (ver 1.0.13.8) package incorporating Mascot search was used for identification and quantification of peptides and proteins. RAW files were searched against a composite target-decoy database containing yeast ORFs from SGD allowing variable modifications of phospho (STY) and oxi (M) and a fixed modification of carbamidomethyl(C) within 7 ppm (MS) and 0.5 Da (MSMS) mass tolerances. Peptide and protein identifications were maintained within 5% FDR.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Hye Kyong Kweon  

PROVIDER: PXD000414 | Pride | 2014-04-16

REPOSITORIES: Pride

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Publications

The yeast Sks1p kinase signaling network regulates pseudohyphal growth and glucose response.

Johnson Cole C   Kweon Hye Kyong HK   Sheidy Daniel D   Shively Christian A CA   Mellacheruvu Dattatreya D   Nesvizhskii Alexey I AI   Andrews Philip C PC   Kumar Anuj A  

PLoS genetics 20140306 3


The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as  ...[more]

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