Modern quantitative mass spectrometry (MS)-based proteomics enables researchers to unravel signaling networks by monitoring proteome-wide cellular responses to different stimuli. MS-based analysis of signaling systems usually requires an integration of multiple quantitative MS experiments, which remains challenging, given that the overlap between these datasets is not necessarily comprehensive. In a previous study we analyzed the impact of the yeast mitogen-activated protein kinase (MAPK) Hog1 on the hyperosmotic stress-affected phosphorylome. Using a combination of a series of hyperosmotic stress and kinase inhibition experiments, we identified a broad range of direct and indirect substrates of the MAPK. Here we re-evaluate this extensive MS dataset and demonstrate that a combined analysis based on two software packages, MaxQuant and Proteome Discoverer, increases the coverage of Hog1 target proteins by 30%. Using protein-protein proximity assays we show that the majority of new targets gained by this analysis are indeed Hog1 interactors. Additionally, kinetic profiles indicate clear-cut and differential trends of Hog1-dependent versus Hog1-independent phosphorylation sites. Our findings highlight a previously unrecognized interconnection between Hog1 signaling and the RAM signaling network, as well as sphingolipid homeostasis.

Peptides were separated using a 60 min gradient (HPLC setup as described above). PRM data acquisition was performed using a scheduled method with 6 min windows for each target based on the retention time determined from a prior data-dependent (DDA) LC-MS/MS run (analyzed using Proteome Discoverer as described above) of 5% mock-treated samples. Raw data were obtained on an Orbitrap QExactive HF-X (Thermo Fisher Scientific) mass spectrometer applying the following settings: survey scan with 30k resolution, AGC 1E6, 30 ms IT, over a range of 380 to 1400 m/z, PRM scan with 30k resolution, AGC 1E5, 100ms IT, isolation window of 0.7 m/z with 0.2 m/z offset, and NCE of 27%. Data analysis, manual validation of all transitions (based on retention time, relative ion intensities, and mass accuracy), and relative quantification was performed in Skyline. Up to six characteristic transitions were selected for each peptide and their peak areas were summed up for peptide quantification across charge states (total peak area). Unphosphorylated peptides that were not affected by any phosphorylation event were considered for normalization of the remaining peptides of the same protein. In a second normalization step peptides were normalized to the respective median (normalized) intensity in the mock experiments (in log10-space).

Hog1as cells expressing Kic1-, Orm2-, and Vps53-HB tandem affinity tag fusion proteins were grown to OD600=1, treated either with DMSO (mock) or 0.25, 0.5, 5µM as-inhibitor SPP86 (Tocris Bioscience) for 5 minutes, followed by a 5 minutes exposure to 0.5M NaCl. All experimental conditions were analysed in triplicates [N(biological replicates)=3]. Samples 1,5 & 9: mock: +DMSO, +0.5M NaCl; Samples 2,6 & 10: +0.2µM SPP86, +0.5M NaCl; Samples 3,7 & 11: +0.5µM SPP86, +0.5M NaCl; Samples 4,8 & 12: +5µM SPP86, +0.5M NaCl. Sample preparation/HB pull downs: Cells were harvested by filtration, deep frozen and grinded using a SPEX Freezer Mill 6870 (SPEX SamplePrep, Metuchen, NJ, USA) applying standard settings. The cell powder was suspended in buffer 1 (6 M guanidine HCl, 50 mM Tris pH8.0, 5 mM NaF, 1 mM PMSF, 0.1% Tween, protease inhibitor cocktail (Roche, Basel, Switzerland, 11 873 580 001), pH 8) and cleared by centrifugation (13500 x g, 15 min, 4°C), incubated with Ni2+-Sepharose beads (GE Healthcare, Buckinghamshire, UK, 17–5318-06) for four hours at room temperature, washed with urea buffer (8M urea, 50mM sodium phosphate buffer pH8.0 (and pH6.3), 300mM NaCl, 0.1% Tween 20). Proteins were eluted in urea buffer pH4.3 containing 10 mM EDTA, incubated with streptavidin-agarose beads, washed with urea wash buffer containing 1% SDS and without SDS. Beads were re-buffered to 50 mM ammonium bicarbonate (ABC). Samples were reduced using DTT, Cys-residues were alkylated with 20mM iodoacetamide (IAA), incubated with 300 ng trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega) at 37°C overnight, quenched with trifluoroacetic acid (0.5% final concentration) and desalted using C18 Stagetips.