Frozen iPSC pellets were thawed and washed with PBS twice prior to lysis. The protein content of the cells was extracted by re-dissolving the pellets in 8 M urea, 100 mM TEAB, pH 8.5 and mixing at room temperature for 15 minutes. Next, the DNA content of the cells was sheared using ultrasonication. The protein amount was determined using a fluorescence based assay (EZQ, Life Technologies) prior to double digestion using mass spectrometry grade lysyl endopeptidase (Wako, Japan) and trypsin (Pierce) in a substrate-to-enzyme ratio of 1:50; w:w, at a final urea concentrations of 2 M and 0.8 M, respectively. The digested proteins were desalted using sepak vacuum cartridges (waters) and dried in vacuo. The desalted peptides were redissolved in (10 mM borate at pH 9.3 : acetonitrile; 80:20; v:v) for hSAX fractionation using a 40 minute gradient. A total of 16 fractions were collected, desalted and dried. The hSAX fractions were redissolved in 5% formic acid for label-free LC-MS analysis. In addition to individual samples, a composite reference sample (‘HPSI_composite_1503’) was constructed by pooling together protein lysates from 43
