Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take centre stage in regulating this process in S. cerevisiae. To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Reduced phosphorylation of Nth1 S83 upon PKA- and Ypk1-inhibition was validated by Western blotting and PRM.

Samples for targeted proteomics by PRM were prepared from 250 µg of protein of yeast cultures (wt, PKA-, Ypk1- or PKA+Ypk1- analog sensitive) treated with 500 nM 1NM-PP1 or DMSO for 15 min. Proteins were digested with LysC and trypsin and a mixture of magnetic TiO2, ZrO2 and Ti(IV)-IMAC beads (15 µl each; ReSyn Biosciences) were used for phospho-peptide enrichment, essentially following the manufacturer`s instructions. Data acquisition was performed on an Orbitrap Fusion Tribrid mass spectrometer coupled to an nLC1000 uHPLC without use of internal standard peptides (Tier 3). Peptides were separated on a 25 cm EASY-spray column (Thermo Scientific) with a 30 min gradient from 3 to 25% acetonitrile, 0.1% formic acid vs. 0.1% formic acid in water. MS-scans were acquired at 60 000 resolution, with an ion target of 4x 105 and maximum injection time of 118 ms. Targeted MS/MS employing higher energy collisional dissociation on 9 selected precursors representing Nth1 phospho-peptides and targeted single ion monitoring on two precursors were each acquired at a resolution of 60 000 with an ion target of 4x 105 and maximum injection time of 200 ms. For data analysis in Skyline 4.2.0.19072 we employed spectral libraries from previously generated data-dependent acquisition and filtered for seven b- or y-ions from ion 3 to last ion.

Three replicates each of 250 µg of protein of yeast cultures (wt, PKA-, Ypk1- or PKA+Ypk1- analog sensitive) treated with 500 nM 1NM-PP1 or DMSO for 15 min.