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).