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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.
Bioconjugate Chemistry
|June 23, 2021
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.
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.
