Quantitative tandem-mass-tag based proteomics analysis and statistical analysis
Methods of proteomics analysis were described in our previous publication (Nightingale et al., 2018), and are briefly described here with a detailed description in the supplementary information. Whole cell lysates were digested into peptides with LysC and trypsin, and equal amounts of peptide labelled with tandem-mass-tag (TMT) reagents. Enriched, labelled peptides were subjected to liquid chromatography coupled with multi-stage mass spectrometry (LC-MS3) prior to quantification of ~2,500 proteins in a single mass spectrometry analysis using an Orbitrap Fusion Lumos (Thermo). To acquire more comprehensive data, TMT-labelled peptide samples were subjected to high pH reversed-phase fractionation (HpRP) to generate 12 combined peptide fractions prior to mass spectrometry. Mass spectra were processed using a SEQUEST-based software pipeline for quantitative proteomics, “MassPike,” through a collaborative arrangement with Professor Steven Gygi’s laboratory at Harvard Medical School. The method of significance A was used to estimate the p-value that each ratio was significantly different to 1 using Perseus version 1.5.1.6 (Cox and Mann, 2008). Values were adjusted for multiple hypothesis testing using the method of Benjamini-Hochberg (Cox and Mann, 2008).