Database search and label-free quantification

The Raw LC-MS/MS data was then analyzed via MaxQuant v1.5.4.1 using Andromeda search employing a target-decoy paradigm to control MS/MS peptide spectral match false discovery rate, and a database of contaminants plus the mouse Uniprot database (n=54,489 protein isoforms, downloaded April 2015). MaxQuant search parameters allowed up to 2 miscleavages, fully tryptic peptides only, and fixed modification of cysteine by carbamidomethylation (+57.0215 Da), plus variable oxidation of methionine (+15.9949 Da) and protein N-terminal acetylation (+42.0106 Da). Other search settings included a maximum peptide mass of 4,600 Da, a minimum peptide length of 7 residues, 0.05 Da tolerance for high resolution MS/MS scans. Co-fragmented peptide search was enabled to deconvolute multiplex spectra. The false discovery rate (FDR) for peptide spectral matches, proteins, and site decoy fraction were all set to 1 percent. The “match between runs” function was used to recover unsequenced precursors. Matches were matched within a 0.7 minute retention time match window and a 20 minute alignment. Quantitation of proteins was performed using LFQ with consideration of only razor and unique peptides. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE²⁴ partner repository with the dataset identifier PXD015202.