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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
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Super-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level
Anton N Kozhinov1, Alex Johnson2,3,4, Konstantin O Nagornov1
1Spectroswiss, 1015 Lausanne, Switzerland.
Analytical Chemistry
|February 7, 2023
Summary
New super-resolution mass spectrometry enables highly multiplexed quantitative proteomics using all TMTpro reporter ion channels. This approach overcomes limitations of current Orbitrap instruments, achieving accurate and precise results for shotgun proteomics.
Area of Science:
- Proteomics
- Mass Spectrometry
- Analytical Chemistry
Background:
- Tandem mass spectrometry (MS2) quantitative proteomics uses reporter ion approaches like TMTc and TMTproC to address ratio compression.
- Current Orbitrap instruments struggle to resolve the high mass resolution (6.32 mDa-spaced) complement reporter ions needed for full multiplexing.
- This limitation restricts TMT/TMTpro reagent usage to fewer channels (5/11 for TMT, 9/18 for TMTpro) with 1 Da spacing.
Purpose of the Study:
- To demonstrate that Orbitrap instruments can resolve 6.32 mDa-spaced complement reporter ions.
- To implement a super-resolution mass spectrometry approach for achieving shotgun proteomics-compatible scan rates.
- To enable highly multiplexed, accurate, and precise quantitative proteomics at the MS2 level.
Main Methods:
- Utilized a Fusion Lumos Orbitrap mass spectrometer with extended 3s transients to resolve complement reporter ions.
- Implemented a super-resolution mass spectrometry approach employing the least-squares fitting (LSF) method on Orbitrap transients.
- Evaluated LSF performance with varying transient durations (108 ms and 256 ms) and Orbitrap resolutions (50,000 and 120,000 at m/z 200).
Main Results:
- Demonstrated resolution of 6.32 mDa-spaced complement reporter ions using extended transients on an Orbitrap.
- LSF processing enabled resolution of all TMTproC channels with transients as short as 108 ms (50,000 resolution).
- Achieved measurement precision comparable to 1 Da spacing using 256 ms transients (120,000 resolution), with minimal loss at 108 ms.
Conclusions:
- The study validates the feasibility of resolving closely spaced complement reporter ions on Orbitrap instruments.
- Super-resolution mass spectrometry with LSF processing allows for full multiplexing of TMTpro reagents in shotgun proteomics.
- This advancement facilitates highly multiplexed, accurate, and precise quantitative proteomics at the MS2 level.
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