biostudies-literatureKozhinov ANPrinceton UniversityEuropean Research CouncilPrinceton Catalysis Initiative, Princeton UniversityBiological and Environmental ResearchH2020 Future and Emerging TechnologiesNational Institute of General Medical SciencesNIGMS NIH HHS3712-3719https://www.ebi.ac.uk/biostudies/studies/S-EPMC9974827biostudies-literatureUnknown95(7)In tandem mass spectrometry (MS2)-based multiplexed quantitative proteomics, the complement reporter ion approaches (TMTc and TMTproC) were developed to eliminate the ratio-compression problem of conventional MS2-level approaches. Resolving all high <i>m</i>/<i>z</i> complement reporter ions (∼6.32 mDa-spaced) requires mass resolution and scan speeds above the performance levels of Orbitrap^TM instruments. Therefore, complement reporter ion quantification with TMT/TMTpro reagents is currently limited to 5 out of 11 (TMT) or 9 out of 18 (TMTpro) channels (∼1 Da spaced). We first demonstrate that a Fusion^TM Lumos^TM Orbitrap can resolve 6.32 mDa-spaced complement reporter ions with standard acquisition modes extended with 3 s transients. We then implemented a sAnalytical chemistrySuper-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level.PMC9974827R35 GM128813R35GM128813DE-SC0018260964553280271Martin WLCorthesy JWuhr MJohnson AKozhinov ANTsybin YOStadlmeier MDayon LNagornov KOfalseSuper-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level.In tandem mass spectrometry (MS2)-based multiplexed quantitative proteomics, the complement reporter ion approaches (TMTc and TMTproC) were developed to eliminate the ratio-compression problem of conventional MS2-level approaches. Resolving all high <i>m</i>/<i>z</i> complement reporter ions (∼6.32 mDa-spaced) requires mass resolution and scan speeds above the performance levels of Orbitrap^TM instruments. Therefore, complement reporter ion quantification with TMT/TMTpro reagents is currently limited to 5 out of 11 (TMT) or 9 out of 18 (TMTpro) channels (∼1 Da spaced). We first demonstrate that a Fusion^TM Lumos^TM Orbitrap can resolve 6.32 mDa-spaced complement reporter ions with standard acquisition modes extended with 3 s transients. We then implemented a s2023-01-01T00:00:00Z2023 Feb2026-03-18T14:13:14.516Z2025-04-04T13:15:41.199ZS-EPMC99748273674992810.1021/acs.analchem.2c04742