Mass spectrometric data were collected on an Orbitrap Fusion Lumos mass spectrometer in-line with a Proxeon NanoLC-1200 UHPLC. The 100 μm capillary column was packed with 35 cm of Accucore 150 resin (2.6 μm, 150Å; ThermoFisher Scientific). Spectra were converted to mzXML using a modified version of ReAdW.exe. Database searching included all entries from the Saccharomyces Genome Database (SGD; August 21, 2017). This database was concatenated with one composed of all protein sequences in the reversed order. Searches were performed using a 50-ppm precursor ion tolerance for total protein level profiling. The product ion tolerance was set to 0.9 Da. These wide mass tolerance windows were chosen to maximize sensitivity in conjunction with SEQUEST searches and linear discriminant analysis (16 (link), 17 (link)). TMT tags on lysine residues and peptide N termini (+229.163 Da) and carbamidomethylation of cysteine residues (+57.021 Da) were set as static modifications, while oxidation of methionine residues (+15.995 Da) was set as a variable modification. For phosphorylation analysis, deamidation (+0.984) on asparagine and glutamine and phosphorylation (+79.966) on serine, threonine, and tyrosine were set as variable modifications. Peptide-spectrum matches (PSMs) were adjusted to a 1% false discovery rate (FDR) (18 (link), 19 (link)). PSM filtering was performed using a linear discriminant analysis, as described previously (17 (link)) and then assembled further to a final protein-level FDR of 1% (19 (link)). Phosphorylation site localization was determined using the AScore algorithm (13 (link)). AScore is a probability-based approach for high-throughput protein phosphorylation site localization. Specifically, a threshold of 13 corresponded to 95% confidence in site localization. Proteins were quantified by summing reporter ion counts across all matching PSMs, as described previously (20 (link)). Reporter ion intensities were adjusted to correct for the isotopic impurities of the different TMT reagents according to manufacturer specifications. The signal-to-noise (S/N) measurements of peptides assigned to each protein were summed and these values were normalized so that the sum of the signal for all proteins in each channel was equivalent, to account for equal protein loading. Lastly, each protein was scaled such that the summed signal-to-noise for that protein across all channels was greater than 100, thereby generating a relative abundance (RA) measurement. A detailed description of the methods in a step-by-step outline is available in the Supplementary Materials.