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Related Experiment Video

Updated: Dec 1, 2025

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
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HYpro16: A Two-Proteome Mixture to Assess Interference in Isobaric Tag-Based Sample Multiplexing Experiments.

Jose Navarrete-Perea1, Steven P Gygi1, Joao A Paulo1

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

Journal of the American Society for Mass Spectrometry
|November 11, 2020
PubMed
Summary

A new HYpro16 standard helps assess isobaric tagging interference in proteome profiling. Real-time database searching (RTS)-MS3 offers the most accurate quantification, improving mass spectrometry performance.

Keywords:
LumosMulti-NotchSPS-MS3TMTmultiplexstandard

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Quantitative Biology

Background:

  • Isobaric tagging is crucial for global proteome profiling.
  • Interference from coeluting peptides can compromise quantitative accuracy in isobaric tagging.
  • A robust standard is needed to evaluate and improve quantification methods.

Purpose of the Study:

  • To develop and validate a two-proteome standard (HYpro16) for assessing interference in isobaric tagging.
  • To evaluate the impact of MS2 isolation window width on quantification accuracy.
  • To compare different data acquisition methods (hrMS2, SPS-MS3, RTS-MS3) for improved accuracy.

Main Methods:

  • Creation of the HYpro16 standard with TMTpro16-labeled human and yeast peptides at varying ratios.
  • Systematic variation of MS2 isolation window width.
  • Acquisition and analysis of data using hrMS2, SPS-MS3, and RTS-MS3 methods.

Main Results:

  • Wider isolation windows increased TMT signal but reduced ratio accuracy.
  • Real-time database searching (RTS)-MS3 yielded the most accurate quantitative ratios.
  • RTS-MS3 quantified nearly as many yeast proteins as hrMS2 when using a yeast-specific database.

Conclusions:

  • The HYpro16 standard effectively challenges mass spectrometers to measure protein abundance ratios under interference.
  • RTS-MS3 is a superior method for accurate quantification in isobaric tagging experiments.
  • This standard enables instrument benchmarking and performance tracking for reliable proteomic analysis.