Proteomics Sample Preparation: Cell pellets (1x10 7 cells) were prepared according to the S-trap protocol. Briefly, cells were lysed in a 10 % (w/v) SDS lysis solution, and sonicated. To remove DNA, 2 µL Benzonase (Cambridge Bioscience Limited, UK) was added, and the samples were sonicated again. Samples were clarified by centrifugation and protein concentration of lysates was determined using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, UK). For each sample 30 µg protein was reduced using TCEP (Cambridge Bioscience Limited, UK) for 15 minutes at 55 C and alkylated using 20 mM IAA (Fluorochem Limited, UK) for 1 hour in the dark at room temperature, then 2.5 µL phosphoric acid (VWR International Ltd, UK) was added. Protein was precipitated through the addition of 165 µL binding/wash buffer and applied to S-Trap™ 20 micro spin columns (VWR International, UK). Sample were washed three times with 150 µL binding/wash buffer, and 1:10 ratio of trypsin gold (Promega, UK) to protein was added to the S-trap and incubated overnight at 37 ˚C in a thermomixer (Starlabs, UK). After incubation, peptides were eluted with 50 µL of the appropriate elution buffers. The eluted solution was dried using an Eppendorf concentrator plus before being resuspended in 0.1 % (v/v) formic acid before MS analysis. Fractionated peptides for spectral library creation were prepared by applying 100 µg peptide mixtures harvested from samples of the same cell line to Pierce™ high pH reversed-phase peptide fractionation spin columns and prepared according to protocol (ThermoFisher Scientific, UK).