High-Throughput Proteomic Analysis with Orbitrap MS

Label-free analyses
of peptides were acquired over 120 min by a Q-Exactive Plus Orbitrap
MS (Thermo Scientific) from 100 ng of peptides (as a proportion of
protein input). TMT-labeled peptide fractions were analyzed over 60
or 120 min by an Orbitrap Eclipse MS (Thermo Scientific) using SPS
MS³ mode. Raw files were processed and analyzed with Proteome
Discoverer 2.5, searching against UniProt Swiss-Prot (version 2021_01,
canonical). Additional analysis was performed in Microsoft Excel.
The MS proteomics data have been deposited to the ProteomeXchange
Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner
repository^{26 (link)} with the data set identifier
PXD032095 and, for validation work, PXD028736 and PXD028768. Proteomics
data are detailed in Tables S1–S20. Annotation enrichment was performed with DAVID and PANTHER. Additional
analyses were performed with CamSol,^{27 (link)} the
PROMPT tool,^{28 (link)} and Proteome-pI.^{29 (link)}