13c315n1 serine

Proteomics analysis was performed as described previously [57] . Briefly, yeast cells were lysed using the filter aided sample preparation method [58] (link). For full proteome analysis, samples were eluted from a PepMap C18 easy spray column (Thermo) with a linear gradient of acetonitrile from 10-35% in H2O with 0.1% formic acid for 118 min at a constant flow rate of 300 nl/min.
For serine incorporation assays using [ 13 C3 15 N1]-serine (Cambridge Isotope Labs) samples were eluted from a PepMap C18 easy spray column (Thermo) with a linear gradient of acetonitrile from 10-50% in 0.1% formic acid for 33 min at a constant flow rate of 300 nl/min.
The resulting MS and MS/MS spectra were analyzed using MaxQuant (version 1.6.0.13, www. maxquant.org/; Cox et al., 2011; (link)Cox and Mann, 2008) (link) as described previously [57] . For incorporation tests, [ 13 C3 15 N1]-serine was added as a modification to the MaxQuant database (mass change =4.0070994066 Da). For the calculation of incorporation rates, the peptide list was filtered for serine containing peptides with a valid heavy/light ratio. For each peptide, the incorporation was calculated as 1 -(1/(ratio H/L -1)). The maximum of a density distribution of all peptides represents the estimated incorporation level. All calculations and plots were performed with the R software package (www.r-project.org/; RRID:SCR_001905).