The composition of the striatin-interacting phosphatases and kinases (STRIPAK) signaling complex is highly similar in all eukaryotes and controls for the phosphorylation and dephosphorylation of target proteins. Associated with STRIPAK are germinal center kinases (GCK), some of which are part of conserved downstream signaling pathways. Using protein samples from wild type and three STRIPAK mutants, we applied absolute quantification by parallel reaction monitoring (PRM) to analyse phosphorylation site occupancies.

Synthesis of all SIS peptides was performed in-house by the use of a Syro I synthesis unit (MultiSynTech, Witten, Germany) and Fmoc chemistry. Samples were analyzed on an Ultimate 3000 nanoRSLC HPLC system coupled to a Q Exactive HF mass spectrometer (MS, both Thermo Scientific). The HPLC was equipped with a trapping column (100 µm x 2 cm C18, PepMap RSLC, Thermo Scientific) for preconcentration and an analytical column (75 µm x 50 cm C18, PepMap RSLC, Thermo Scientific) for separation of the peptides. In order to verify the linearity of response and to determine the limit of blank (LOB), limit of detection (LOD), and lower limit of quantification (LLOQ), response curves of both phosphorylated and non-phosphorylated SIS peptides were acquired. In both cases, a background matrix was generated by pooling aliquots of all individual samples. For the analysis of the individual samples, a total amount of non-phosphorylated SIS peptides ranging from 0.05 fmol to 60 fmol was spiked into 3 µg of total protein digest, depending on the expected endogenous concentration. For the phosphopeptides, a total amount of 410 amol to 4.1 fmol was spiked into 450 µg of protein digest, followed by phosphopeptide enrichment and nano-LC-MS/MS measurement of 25 % of the eluates after enrichment.

S. macrospora strains, were grown under standard conditions and transformed with recombinant plasmids as described before (Engh et al. 2007, Dirschnabel et al. 2014). S. macrospora strains were precultured in Petri dishes with 20 ml liquid BMM with two biological replicates per strain at 27°C for 2 days. Three standardized inoculates of each BMM preculture were transferred in Petri dishes with 20 ml liquid BMM and grown at 27°C and 40 rpm for 3 days. For cell wall lysis and protein extraction, mycelium was harvested and freezed within 60 s. The frozen mycelium was ground in liquid nitrogen and suspended in FLAG extraction buffer. Afterward, the samples were centrifuged at 4°C and 15,000 rpm for 30 min.