on 06-17-201603:22 AM - edited on 10-15-202112:10 PM by AnalyteGuru
Robert Cunningham1; Kratika Singhal1; Ryan T Fellers2; Luca Fornelli2; Henrique Dos Santos Seckler2; Bhavin Patel1; Egle Capkauske3; Juozas Siurkus3; Philip Compton2; Neil L Kelleher2; John C Rogers1
Purpose To synthesize two active forms of recombinant methionine sulfoxide reductase (MetSR) enzymes and perform proof of principle studies for mass spectrometry applications in intact protein, targeted quantitative peptides, and shotgun proteomics.
Methods Two recombinant MetSR and a methionine-rich protein were expressed, purified, and tested for gel mobility shifts prior to use in MS applications. For intact and shotgun proteomics experiments ,hydrogen peroxide was added to induce mild methionine oxidation and compared to non-oxidized controls before and after treatment with MetSR. Reduction was carried out using DTT as a cofactor and samples were analyzed by a Thermo Scientific™ Orbitrap XL™ Hybrid Ion Trap-Orbitrap MS . Targeted quantitative analysis of methionine containing peptide and phosphopeptides were done on a Thermo Scientific™ Q Exactive™ HF MS using Parallel Reaction Monitoring.
Results: We successfully produced and purified two active MetSR enzymes, as observed by gel shift of an oxidized methionine rich protein, reduction of oxidized methionines in an intact protein, and in shotgun and targeted proteomics experiments at the peptide level. Parallel Reaction Monitoring was used to demonstrate 90-99% reduction in oxidized methionine in MetSR treated peptide and phosphopeptides.
1Thermo Fisher Scientific, Rockford, IL
2Northwestern University, Evanston, IL
3Thermo Fisher Scientific, Vilnius, Lithuania