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. 2018 Apr 1;162(2):429-438.
doi: 10.1093/toxsci/kfx265.

Determination of Protein Haptenation by Chemical Sensitizers Within the Complexity of the Human Skin Proteome

Erika Parkinson  1   2 , Maja Aleksic  3 , Richard Cubberley  3 , Gushinder Kaur-Atwal  4 , Johannes P C Vissers  4 , Paul Skipp  1   2
Affiliations

Determination of Protein Haptenation by Chemical Sensitizers Within the Complexity of the Human Skin Proteome

Erika Parkinson et al. Toxicol Sci. .

Abstract

Skin sensitization associated with the development of allergic contact dermatitis occurs via a number of specific key events at the cellular level. The molecular initiating event (MIE), the first in the sequence of these events, occurs after exposure of the skin to an electrophilic chemical, causing the irreversible haptenation of proteins within skin. Characterization of this MIE is a key step in elucidating the skin sensitization adverse outcome pathway and is essential to providing parameters for mathematical models to predict the capacity of a chemical to cause sensitization. As a first step to addressing this challenge, we have exposed complex protein lysates from a keratinocyte cell line and human skin tissue with a range of well characterized sensitizers, including dinitrochlorobenzene, 5-chloro-2-methylisothiazol-3-one, cinnamaldehyde, and the non (or weak) sensitizer 6-methyl coumarin. Using a novel stable isotope labeling approach combined with ion mobility-assisted data independent mass spectrometry (HDMSE), we have characterized the haptenome for these sensitizers. Although a significant proportion of highly abundant proteins were haptenated, we also observed the haptenation of low abundant proteins by all 3 of the chemical sensitizers tested, indicating that within a complex protein background, protein abundance is not the sole determinant driving haptenation, highlighting a relationship to tertiary protein structure and the amino acid specificity of these chemical sensitizers and sensitizer potency.

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Figures

Figure 1.
Figure 1.
Distribution of ion intensity between unmodified and sensitizer modified peptides. Modified peptides are observed at lower ion intensity compared with unmodified peptides within the same sample.
Figure 2.
Figure 2.
Graphical representation of all the modified proteins identified with the keratinocyte cell (A) and ex vivo skin (B) lysates, ranked by their abundance which was based on their estimated protein concentration (fmol). The x-axis represents all of the proteins identified in each lysate from most abundant to least; the y-axis shows which of those proteins were modified by each of the 3 chemical sensitizers tested in this study.
Figure 3.
Figure 3.
Circular representation of the nucleophilic concentration of each protein identified within the keratinocyte (A) and ex vivo skin (B) lysates, from highest concentration moving clockwise to the lowest concentration of nucleophiles. Proteins that were modified by each of the adducts observed in this study are highlighted within these circular representations, to show the distribution of nucleophile concentration amongst the haptenated proteins.
Figure 4.
Figure 4.
Percentage of available nucleophilic residues haptenated by adducts of DNCB, MCI, and CA in the keratinocyte (A) and ex vivo skin (B) lysates. Light Gray bars indicate where the exact site of sensitizer modification could not be confirmed from the fragmentation spectra.
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References

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