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

Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers
Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers
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-MS(E) 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-MS(E) 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
1096-6080
239-249
Parkinson, E.
29a3198f-3d30-4a98-b4ab-e9ec44db68fa
Boyd, P.
509f4035-a6d0-40e0-9a48-12836a96fee3
Aleksic, M.
50a79aa9-e4b9-41c2-ae34-30044933aae8
Cubberley, R.
2c672559-9b75-44a7-ae2a-4aa60eba8d2f
O'Connor, D.
c4910888-1df5-4be7-8142-af0e919f9100
Skipp, P.
1ba7dcf6-9fe7-4b5c-a9d0-e32ed7f42aa5
Parkinson, E.
29a3198f-3d30-4a98-b4ab-e9ec44db68fa
Boyd, P.
509f4035-a6d0-40e0-9a48-12836a96fee3
Aleksic, M.
50a79aa9-e4b9-41c2-ae34-30044933aae8
Cubberley, R.
2c672559-9b75-44a7-ae2a-4aa60eba8d2f
O'Connor, D.
c4910888-1df5-4be7-8142-af0e919f9100
Skipp, P.
1ba7dcf6-9fe7-4b5c-a9d0-e32ed7f42aa5

Parkinson, E., Boyd, P., Aleksic, M., Cubberley, R., O'Connor, D. and Skipp, P. (2014) Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers. Toxicological Sciences, 142 (1), 239-249. (doi:10.1093/toxsci/kfu168). (PMID:25145658)

Record type: Article

Abstract

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-MS(E) 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-MS(E) 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

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e-pub ahead of print date: 21 August 2014
Organisations: Centre for Biological Sciences

Identifiers

Local EPrints ID: 377769
URI: http://eprints.soton.ac.uk/id/eprint/377769
ISSN: 1096-6080
PURE UUID: 37c10a18-6579-4f37-8cb7-4a66d663668f
ORCID for P. Skipp: ORCID iD orcid.org/0000-0002-2995-2959

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Date deposited: 18 Jun 2015 11:00
Last modified: 26 Nov 2019 02:04

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