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Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT
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Isotope-Coded Maleimide Affinity Tags for Proteomics Applications.

Adam P Wdowiak, Marisa N Duong1, Rohan D Joyce

  • 1Proteomics International, Perth, 6009, Australia.

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|June 23, 2021
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Summary

New isotope-coded maleimide affinity tags (ICMATs) improve thiol-selective labeling for mass spectrometry. A phenylene linker enhances compatibility with proteomics workflows, overcoming limitations of previous ICATs.

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Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling
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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Isotope-coded affinity tags (ICATs) are essential for quantitative proteomics, particularly for analyzing cysteine thiol oxidation states in biological samples.
  • Commercial ICATs often use iodoacetamido groups, which exhibit limited thiol selectivity and slow reaction rates, potentially yielding inaccurate results.
  • There is a need for improved affinity tags that offer enhanced selectivity and efficiency for thiol labeling in proteomics.

Purpose of the Study:

  • To design and synthesize novel isotope-coded affinity tags (ICMATs) utilizing thiol-selective N-alkylmaleimide electrophiles.
  • To evaluate ICMATs as mass spectrometry probes for the ratiometric analysis of protein thiol oxidation states.
  • To optimize ICMATs for compatibility with standard proteomics workflows, including HPLC separation.

Main Methods:

  • Synthesis of three distinct ICMATs featuring N-alkylmaleimide electrophiles with different linker chemistries (butylene and phenylene).
  • Assessment of ICMATs for thiol-selective labeling of lysozyme and complex muscle proteomes.
  • Evaluation of mass spectrometry-based ratiometric analysis and HPLC coelution behavior of ICMAT-labeled peptides.

Main Results:

  • Two ICMAT pairs with butylene/D8-butylene linkers demonstrated effectiveness as mass spectrometry probes but showed poor HPLC coelution.
  • A modified ICMAT design incorporating a phenylene/13C6-phenylene linker resolved the HPLC coelution issue.
  • The phenylene-linked ICMATs maintained efficient adduct formation and thiol-selectivity without compromising quantitative accuracy.

Conclusions:

  • ICMATs with N-alkylmaleimide electrophiles represent a significant improvement over traditional ICATs for thiol-based quantitative proteomics.
  • The phenylene/13C6-phenylene linker is crucial for ensuring compatibility with HPLC-based proteomics workflows.
  • Optimized ICMATs provide a robust and reliable tool for precise analysis of protein thiol oxidation states in various biological contexts.