Peptide Quantification and Identification Workflow

Samples were cleaned using reversed phase liquid chromatography as previously described^{34 (link)} (high pH RP, Agilent 1200 series micro-flow pump, Water XBridge column C18 3.5um, 2.1×100mm). The samples were dried (speed vacuum) and stored at −80 °C until quantification using LavaPep’s Fluorescent Peptide and Protein Quantification Kit (Gel Company) according to manufacturer’s protocol. Equal amounts of peptides for each sample were re-suspended (2% acetonitrile, 0.1% formic acid) and injected into a nano flow liquid chromatography system (Easy nLC, Thermo Fisher) connected inline to a Q Exactive Quadrupole Orbitrap mass spectrometer. Raw MS spectra were processed by Progenesis (Nonlinear Dynamics) and Mascot (Matrix Science) as previously described^{35 (link), 36 (link)}. Technical variability was determined by the addition of a protein mix of all the samples that was injected in 10 sample intervals. Search results were entered into Scaffold (v4.4.1.1; Proteome Software, Portland, OR) to determine protein identifications (80% peptide confidence; 95% protein confidence, with minimum of 2 unique peptides per protein).