We showcased our method using a TMT10-plex of yeast (S. cerevisiae wild-type strain BY4716) grown in synthetic complete media supplemented with 2% glucose (n=5) or 2% pyruvate (n=5) as the carbon source. We harvested the cells at OD600nm=0.8. Cells were lysed by bead-beating in 8 M urea 200mM EPPS (4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid), pH 8.5 and with protease and phosphatase inhibitors. Protein concentration was determined with the BCA assay. The BCA assay was performed according to manufacturer’s instructions with samples that were diluted at least 1:20, to ensure that the 8M urea has been diluted far below its compatibility limit. Samples were reduced with 5mM TCEP, alkylated with 10 mM iodoacetamide that was quenched with 10 mM DTT. A total of 100 μg of protein were chloroform-methanol precipitated. Protein was reconstituted in 200 mM EPPS pH 8.5 and digested by Lys-C overnight and trypsin for 6 h, both at a 1:100 protease-to-peptide ratio. Directly to the digest, we added a final volume of 30% acetonitrile and labelled 100 μg of peptide with 200 μg of TMT. To check mixing ratios, 2 μg of each sample were pooled, desalted, and analyzed by mass spectrometry. Using normalization factors calculated from this “label check,” samples were mixed 1:1 across all channels and desalted using a 100 mg Sep-Pak solid phase extraction column. The Pierce High-Select Fe-NTA Phosphopeptide Enrichment Kit was used to enrich phosphopeptides from the pooled TMT-labeled mixture. The unbound fraction and washes from this enrichment were combined and fractionated with basic pH reversed-phase (BPRP) HPLC, collected in a 96-well plate and combined down to 12 fractions prior to desalting and subsequent LC-MS/MS processing (14 (link), 15 ).