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

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 TiO(2) 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.

MAPK Hog1 closes the S. cerevisiae glycerol channel Fps1 by phosphorylating and displacing its positive regulators

“…These phosphorylations are partly dependent on the MAPK Hog1, suggesting a direct regulatory role of the kinase in Rgc2 function, although Hog1-independent phosphorylation of Rgc2 has also been observed during logarithmic growth as well as in response to hypo-and hyperosmotic stress (Beese et al 2009;Reiter et al 2012). Currently, 19 phosphorylation sites of Rgc2 are documented in the PhosphoPep (part of the Saccharomyces Genome Database) and PhosphoGRID databases (King et al 2006;Stark et al 2010), two of which (phosphoserines 344 and 1021) lie within S/T-P MAPK consensus motifs.…”

“…To unravel the phosphorylation patterns of Rgc2, we conducted MS analysis based on tandem affinity purification, as described (Reiter et al 2012). In total, our analysis of tryptic and chymotryptic digests covered 65.5% of the Rgc2 protein sequence.…”

“…Although Hog1 plays a role in glycerol retention by control of Fps1, the mechanism by which it influences Fps1 activity is unknown. Hog1 function has been mainly associated with the regulation of transcriptional events Rep et al 1999;de Nadal and Posas 2010;de Nadal et al 2011;Saito and Posas 2012), although a recent quantitative mass spectrometry (MS) analysis has revealed several new candidate substrates of Hog1 (Reiter et al 2012). …”

Genotoxic Stress Prevents Ndd1-Dependent Transcriptional Activation of G2/M-Specific Genes in Saccharomyces cerevisiae

“…Integration of NDD1, wild type or mutant, at the LEU locus was done by transforming the respective plasmids after linearization with the ClaI restriction enzyme. An NDD1-HTBeaq strain was obtained by transforming the PCR-amplified HTBeaq cassette as described previously (25). In all cases, positive clones were confirmed by PCR-based analysis and/or Western blotting.…”

Discordance between protein and transcript levels detected by selected reaction monitoring

“…Furthermore, SRM analysis allows the quantification of absolute abundances of posttranslational modifications such as phosphorylation states. [25][26][27] Although less common in the field of plant science, this method also has the potential to be used for other purposes, such as the quantification of stoichiometric changes in protein complexes dependent on environmental stimuli, or the quantification of developmental stages.…”