Each sample was analyzed on a Thermo Scientific Orbitrap Fusion Lumos MS via 3 technical replicate injections using data-dependent acquisition (DDA) HCD MS2 instrument method outlined in Table S1. The technical replicates were blocked, and each block was randomized. Pooled QCs, which are a mixture of all the samples being analyzed, were analyzed at the start, end, and in between each sample block. MS data were analyzed using Proteome Discoverer 2.4 (Thermo Fisher Scientific, Waltham, MA) platform, as outlined in Table S2. Protein identifications were filtered to include only those proteins identified by two or more unique peptides identified.
Differential expression calling was performed on normalized read count data generated by Proteome Discoverer 2.4 (Thermo Fisher Scientific, Waltham, MA) using integrated Differential Expression and Pathway (iDEP) analysis (http://bioinformatics.sdstate.edu/idep/) tool.28 (link) Box plots for data normalization and sample variance were assessed as part of quality control (Supplementary Figure 2). Gene enrichment analysis was subsequently performed on differentially expressed gene lists using ShinyGO 0.76 (http://bioinformatics.sdstate.edu/go/) to identify altered biological pathways between groups.