Super-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level. Author Anton Kozhinov, Alex Johnson, Konstantin Nagornov, Michael Stadlmeier, Warham Martin, Loïc Dayon, John Corthésy, Martin Wühr, Yury Tsybin Publication Year 2023 Type Journal Article Abstract 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 / complement reporter ions (∼6.32 mDa-spaced) requires mass resolution and scan speeds above the performance levels of Orbitrap 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 Lumos Orbitrap can resolve 6.32 mDa-spaced complement reporter ions with standard acquisition modes extended with 3 s transients. We then implemented a super-resolution mass spectrometry approach using the least-squares fitting (LSF) method for processing Orbitrap transients to achieve shotgun proteomics-compatible scan rates. The LSF performance resolves the 6.32 mDa doublets for all TMTproC channels in the standard mass range with transients as short as ∼108 ms (Orbitrap resolution setting of 50,000 at / 200). However, we observe a slight decrease in measurement precision compared to 1 Da spacing with the 108 ms transients. With 256 ms transients (resolution of 120,000 at / 200), coefficients of variation are essentially indistinguishable from 1 Da samples. We thus demonstrate the feasibility of highly multiplexed, accurate, and precise shotgun proteomics at the MS2 level. Keywords Proteomics, Tandem Mass Spectrometry, Ions, Indicators and Reagents Journal Anal Chem Volume 95 Issue 7 Pages 3712-3719 Date Published 2023/02/21 ISSN Number 1520-6882 DOI 10.1021/acs.analchem.2c04742 Alternate Journal Anal Chem PMCID PMC9974827 PMID 36749928 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML