TY - JOUR
T1 - Super-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level
AU - Kozhinov, Anton N.
AU - Johnson, Alex
AU - Nagornov, Konstantin O.
AU - Stadlmeier, Michael
AU - Martin, Warham Lance
AU - Dayon, Loïc
AU - Corthésy, John
AU - Wühr, Martin
AU - Tsybin, Yury O.
N1 - Funding Information:
The authors thank Dr. Tagir Aushev for discussions and are grateful for financial support through the European Research Council under grant agreement #280271 (SRMS4HESUS) and the European Horizon 2020 research and innovation program under grant agreement #964553 (ARIADNE). This work was supported by NIH grant R35GM128813, Princeton Catalysis Initiative, Eric and Wendy Schmidt Transformative Technology Fund, the U.S. Department of Energy, Office of Science, and Office of Biological and Environmental Research under award number DE-SC0018260.
Publisher Copyright:
© 2023 American Chemical Society.
PY - 2022
Y1 - 2022
N2 - 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 OrbitrapTM 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 FusionTM LumosTM 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 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.
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 OrbitrapTM 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 FusionTM LumosTM 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 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.
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U2 - 10.1021/acs.analchem.2c04742
DO - 10.1021/acs.analchem.2c04742
M3 - Article
C2 - 36749928
AN - SCOPUS:85147981777
SN - 0003-2700
JO - Analytical Chemistry
JF - Analytical Chemistry
ER -