Isotope-Coded Maleimide Affinity Tags for Proteomics Applications

Bioconjug Chem. 2021 Aug 18;32(8):1652-1666. doi: 10.1021/acs.bioconjchem.1c00206. Epub 2021 Jun 23.


Isotope-coded affinity tags (ICATs) are valuable tools for mass spectrometry-based quantitative proteomics, in particular, for comparison of protein (cysteine-residue) thiol oxidation state in normal, stressed, and diseased tissue. However, the iodoacetamido electrophile used in most commercial ICATs suffers from poor thiol-selectivity and modest rates of adduct formation, which can lead to spurious results. Hence, we designed and synthesized three ICATs containing thiol-selective N-alkylmaleimide electrophiles (isotope-coded maleimide affinity tags = ICMATs) and assessed these as mass spectrometry probes for ratiometric analysis of lysozyme and muscle proteomes. Two ICMAT pairs containing butylene/D8-butylene linkers were effective MS probes, but not ideal for typical proteomics workflows, because peptides bearing these tags frequently did not coelute with HPLC. A switch to a phenylene/13C6-phenylene linker solved this issue without compromising the efficiency of adduct formation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carbon Isotopes / chemistry*
  • Chromatography, Liquid
  • Dogs
  • Gene Expression Regulation
  • Isotope Labeling / methods*
  • Male
  • Maleimides / chemistry*
  • Mice
  • Mice, Inbred mdx
  • Models, Molecular
  • Muscle Proteins / chemistry
  • Muscle Proteins / genetics
  • Muscle Proteins / metabolism*
  • Muscle, Skeletal
  • Protein Conformation
  • Proteomics / methods*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Tandem Mass Spectrometry


  • Carbon Isotopes
  • Maleimides
  • Muscle Proteins
  • maleimide