The goal of the SC COBRE in Oxidants, Redox Balance and Signaling Proteomics Core is to provide state-of-the-art LC-MS/MS instrumentation, expertise, and training for comprehensive proteomic analyses to advance the research endeavors and career development of junior investigators with interests in redox signaling. Dedicated core personnel assist with experimental design, method development, data acquisition, and computational analysis for protein characterization and quantitative proteomic applications using metabolic labeling (SILAC), isobaric tandem mass tagging (TMT), and label free quantitation (MaxQuant LFQ). The core has facilitated characterization of redox- and drug-sensitive post-translational modifications of cysteine (sulfenic, sulfinic, sulfonic acid, S-glutathionylation, disulfide bonds); arginine (glycation by methylglyoxal, dihydroxyimidazolidine); tyrosine (nitrated and crosslinked), and lysine (acetylation, ubiquitin). COBRE investigators have sequenced putative biomarker peptides discovered by MALDI-tissue imaging mass spectrometry and identified differentially expressed or post-translationally regulated proteins following genetic manipulation of anti-oxidant enzymes, drug treatment, or disease. Quantitative proteomic methodology has been established to determine the effects of altered redox-balance on differential protein abundance in FAC-sorted cell populations, primary cells, and exosomes. COBRE investigators have also utilized quantitative proteomics to elucidate the mechanism of action of HDAC inhibitors and identify the targets of drugs identified in phenotypic screens.
With the recent acquisition of a ThermoScientific Fusion Lumos Orbitrap ETD/UVPD MS, improvements in quantitative proteomics employing tandem mass tagging (TMT SPS-MS3), metabolic labeling (NeuCode SILAC), and label free proteomics (Boxcar) will be available. New complementary peptide fragmentation modes are available (EThcD, UVPD) to facilitate confident sequencing of challenging post-translational modifications. Integration of novel data acquisition modes will facilitate much needed global, targeted proteomics for verification of endogenously post-translationally modified peptides in vivo (MaxQuant.Live). The core has also acquired a Xevo TQ-S triple quadrupole MS which will expand our capabilities to include absolute quantitation and multiple reaction monitoring (MRM) assays for targeted proteomics.
In addition to these new discovery and targeted proteomic capabilities, the core is currently optimizing redox proteomics approaches for selective-reduction and enrichment of differentially modified cysteine residues. We will continue to assist COBRE investigators and members of the COBRE Redox Center with customized method development as needed to advance their research endeavors.Reminder: Please acknowledge NIH support of the Proteomics Core (P20 GM103542-Proteomics Core) and shared instrumentation grants (S10 OD010731-Orbitrap Elite ETD MS awarded in 2012 and S10 OD025126 Orbitrap Fusion Lumos ETD/UVPD MS awarded in 2018).