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Updated: Jun 11, 2025

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Achieving a 35-Plex Tandem Mass Tag Reagent Set through Deuterium Incorporation.

Nathan R Zuniga1, Dustin C Frost2, Karsten Kuhn3

  • 1Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.

Journal of Proteome Research
|October 9, 2024
PubMed
Summary

We enhanced TMTpro reagent multiplexing to 35-plex using deuterium. A novel strategy (DISAT) corrects for deuterium effects, enabling accurate proteomic quantification in cell lines and chemoproteomics.

Keywords:
ABPPAstralTMTproTMTproDdeuteriumisobaric tagging

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Mass spectrometry-based sample multiplexing with isobaric tags enables high-throughput quantitative biological assays.
  • The TMTpro reagent set allows for multiplexed proteomic analysis.
  • Incorporating deuterium into isobaric tags can increase multiplexing but may affect quantification accuracy.

Purpose of the Study:

  • To nearly double the multiplexing capability of the TMTpro reagent set to 35-plex.
  • To develop a strategy to counteract the suboptimal peak coelution caused by deuterium incorporation.
  • To validate the new multiplexing strategy in biological and chemoproteomics experiments.

Main Methods:

  • Incorporation of one deuterium isotope into the reporter group of TMTpro reagents.
  • Development of the "design independent sub-plexes but acquire together" (DISAT) strategy for normalization.
  • Segregation of non-deuterium and deuterium-containing channels into distinct subplexes with reassembly via a common bridge channel.
  • Application of the DISAT strategy in comparative proteomics of human cell lines and a chemoproteomics experiment targeting Pin1.

Main Results:

  • Achieved a 35-plex multiplexing capability for TMTpro reagents.
  • The DISAT strategy effectively compensated for deuterium-induced quantification errors.
  • Demonstrated successful application of the method in comparing protein expression differences between human cell lines.
  • Identified compounds binding to cysteine-113 of Pin1 using the developed chemoproteomics approach.

Conclusions:

  • The modified TMTpro reagent set and DISAT strategy significantly enhance multiplexing capacity for mass spectrometry-based proteomics.
  • This approach maintains high accuracy in quantitative measurements, even with deuterium incorporation.
  • The validated method provides a powerful tool for high-throughput proteome-wide studies and targeted chemoproteomics.