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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
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Boosting the Sensitivity of Quantitative Single-Cell Proteomics with Infrared-Tandem Mass Tags
Trenton M Peters-Clarke1,2, Yiran Liang3, Keaton L Mertz1,2
1Department of Chemistry, University of Wisconsin─Madison, Madison, Wisconsin 53706, United States.
Journal of Proteome Research
|May 7, 2024
Summary
Infrared-tandem mass tags (IR-TMT) enhance single-cell proteomics by maximizing reporter ion generation. This novel method improves quantitative accuracy and throughput for analyzing cellular heterogeneity.
Area of Science:
- Proteomics
- Mass Spectrometry
- Cellular Biology
Background:
- Single-cell proteomics requires high analytical sensitivity due to limited starting material.
- Isobaric tagging methods in proteomics face limitations in quantitative accuracy and precision caused by restricted reporter ion generation.
- Conventional methods like HCD (Higher-energy Collisional Dissociation) exhibit m/z dependence, impacting ion efficiency.
Purpose of the Study:
- To develop a method that maximizes reporter ion generation for improved quantitative accuracy in single-cell proteomics.
- To overcome the limitations of existing mass spectrometry techniques in handling limited sample amounts.
- To enhance the sensitivity and throughput of single-cell proteomic analyses.
Main Methods:
- Development of infrared-tandem mass tags (IR-TMT) combining infrared photoactivation and ion parking.
- Application of IR-TMT to single-cell human proteomes using 18-plex TMTpro.
- Comparative analysis against conventional SPS-MS3 approaches.
Main Results:
- IR-TMT achieved 4-5-fold increases in reporter signal compared to conventional SPS-MS3.
- The method demonstrated 4-5-fold lower injection times, indicating superior sensitivity.
- IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification.
Conclusions:
- IR-TMT significantly enhances the dynamic range and sensitivity of proteomic experiments, particularly for single-cell applications.
- This technique overcomes m/z dependence and reporter degradation issues inherent in other methods.
- IR-TMT is compatible with advanced techniques like gas-phase fractionation and real-time searching, promising broader applications in studying cellular heterogeneity.
