PT  - JOURNAL ARTICLE
AU  - Anton N. Kozhinov
AU  - Alex Johnson
AU  - Konstantin O. Nagornov
AU  - Michael Stadlmeier
AU  - Warham Lance Martin
AU  - Loïc Dayon
AU  - John Corthésy
AU  - Martin Wühr
AU  - Yury O. Tsybin
TI  - Super-resolution mass spectrometry enables rapid, accurate, and highly-multiplexed proteomics at the MS2-level
AID  - 10.1101/2022.07.29.501912
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2022.07.29.501912
4099  - http://biorxiv.org/content/early/2023/01/13/2022.07.29.501912.short
4100  - http://biorxiv.org/content/early/2023/01/13/2022.07.29.501912.full
AB  - 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 m/z 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-second 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 m/z 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 m/z 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.Competing Interest StatementDr. Tsybin, Dr. Nagornov, and Dr. Kozhinov are employees of Spectroswiss, which develops hardware and software tools for mass spectrometry data acquisition and processing. Dr. Dayon and Mr. Corthesy are employees of the Societe des Produits Nestle SA.