Using the global SILAC and label-free phosphoproteomics experiments as a basis, an inclusion list for validation of known and novel AKT, RSK and S6K targets comprising the RxRxxp[ST] motif was generated. Samples were analyzed by PRM-MS.

Eluates from EasyPhos and SMOAC phosphopeptide enrichment were desalted and pooled for targeted MS analysis. To this end, an inclusion list was generated with Skyline daily (version 20.2.1.384; MacLean et al., Bioinformatics, 2010) comprising 391 phosphopeptides (506 precursor ions) containing the RxRxxp[ST] motif. Furthermore, this list contained the 11 peptides from the Biognosys iRT standard and 16 internal control phosphopeptides which were chosen according to the label-free phosphoproteomics experiment. After database search using MaxQuant (version 1.6.10.43) and a murine UniProt sequence database (June 2020, 63,722 entries), the inclusion list, the MaxQuant results file msms.txt (filtered for the PRM raw files), all PRM raw files and the fasta file of the UniProt ProteomeSet for mouse (June 2020) were imported into Skyline. Peptide settings were as follows: protease, trypsin/P; max. missed cleavages, two; time window, 5 min; peptide length, 8-25 amino acids, modifications, C carbamidomethylation, M oxidation and S/T/Y phosphorylation; max. variable modifications, three; max. neutral loss, one. Default orbitrap transition settings were applied. Because of an unstable electrospray, the following experiments were excluded from further data evaluation: IGF replicate (R) 9, MK R4 and PF R5. Peptides with low-quality, interfered MS1 and/or number of MS2 < 4 were removed manually. For phosphopeptides with multiply precursor ions, the one with the highest intensity and most MS2 was kept. Extracted ion chromatograms were inspected for correct peak picking and peak integrations were adjusted if necessary.

C2 myoblasts were grown in DMEM GlutaMAX with 15% FCS, 2% sodium pyruvate and 1% NEAA. Myoblasts were differentiated into myotubes, serum-starved and treated with EPS for 3 h. Myotubes were stimulated with IGF-1 (10 ng/ml) for 30 min or EGF (10 ng/ml) for 15 min, whereas and MK-2206 (10 µM, 30 min), PF-4708671 (10 µM, 30 min) and BI-D1870 (10 µM, 15 min) were used for AKT, S6K and RSK inhibition, respectively. Cell lysis, digestion and phosphopeptide enrichment was performed according to the EasyPhos protocol (Humphrey et al., Nat. Protoc., 2018). In brief, cells were lysed with 4% sodium deoxycholate in 100 mM Tris- buffer, pH 8.5, proteins were subjected to reduction and alkylation, followed by digestion using trypsin and LysC (enzyme:protein ratio, 1:100). Phosphopeptides were enriched using TiO 2 and supernatants obeyed after enrichment were subjected to Fe(III)-IMAC-based phosphopeptide enrichment using SMOAC (Choi et al., at 65th ASMS Conference on Mass Spectrometry and Allied Topics, 2017).