Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers

Erika Parkinson*, Pete Boyd, Maja Aleksic, Richard Cubberley, David O'Connor, Paul Skipp

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

23 Citations (Scopus)


The risk of contact sensitization is a major consideration in the development of new formulations for personal care products. However, developing a mechanistic approach for non-animal risk assessment requires further understanding of haptenation of skin proteins by sensitizing chemicals, which is the molecular initiating event causative of skin sensitization. The non-stoichiometric nature of protein haptenation results in relatively low levels of modification, often of low abundant proteins, presenting a major challenge for their assignment in complex biological matrices such as skin. Instrumental advances over the last few years have led to a considerable increase in sensitivity of mass spectrometry (MS) techniques. We have combined these advancements with a novel dual-labeling/LC-MSE approach to provide an in-depth direct comparison of human serum albumin (HSA), 2,4-dinitro-1-chlorobenzene (DNCB), 5-chloro-2-methyl-4-isothiazolin-3-one (MCI), trans-cinnamaldehyde, and 6-methyl coumarin. These data have revealed novel insights into the differences in protein haptenation between sensitizers with different reaction mechanisms and sensitizing potency; the extreme sensitizers DNCB and MCI were shown to modify a greater number of nucleophilic sites than the moderate sensitizer cinnamaldehyde; and the weak/non-sensitizer 6-methyl coumarin was restricted to only a single nucleophilic residue within HSA. The evaluation of this dual labeling/LC-MSE approach using HSA as a model protein has also demonstrated that this strategy could be applied to studying global haptenation in complex mixtures of skin-related proteins by different chemicals.

Original languageEnglish
Pages (from-to)239-249
Number of pages11
JournalToxicological Sciences
Issue number1
Publication statusPublished - 1 Nov 2014


  • Contact allergy
  • GeLC-MS/MS
  • Proteomics
  • Sensitizer
  • Skin

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