title: Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers.
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.
authors.affiliation: Centre for Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK Centre for Proteomic Research, Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, UK epp@soton.ac.uk.
authors.forename: Erika
authors.initials: E
authors.lastname: Parkinson
authors.affiliation: Centre for Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK Centre for Proteomic Research, Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, UK.
authors.forename: Pete
authors.initials: P
authors.lastname: Boyd
authors.affiliation: Safety and Environmental Assurance Centre, Unilever, Colworth Science Park, Sharnbrook, MK44 1LQ, UK.
authors.forename: Maja
authors.initials: M
authors.lastname: Aleksic
authors.affiliation: Safety and Environmental Assurance Centre, Unilever, Colworth Science Park, Sharnbrook, MK44 1LQ, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Cubberley
authors.affiliation: Department of Biological Sciences, Xi'an Jiaotong-Liverpool University, SIP Suzhou, Jiangsu Province 215123, China.
authors.forename: David
authors.initials: D
authors.lastname: O'Connor
authors.affiliation: Centre for Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK Centre for Proteomic Research, Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Skipp
mesh_terms: D000171:Acrolein / Q000031:analogs & derivatives* / Q000737:chemistry; D002853:Chromatography, Liquid; D003374:Coumarins / Q000737:chemistry*; D003877:Dermatitis, Contact / Q000378:metabolism*; D004137:Dinitrochlorobenzene / Q000737:chemistry*; D006241:Haptens / Q000737:chemistry*; D006801:Humans; D007553:Isotope Labeling; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry*; D012867:Skin / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013844:Thiazoles / Q000737:chemistry*
chemical_list: D003374:Coumarins; D004137:Dinitrochlorobenzene; D006241:Haptens; D012709:Serum Albumin; D013844:Thiazoles; D000171:Acrolein; C011421:5-chloro-2-methyl-4-isothiazolin-3-one; C018096:6-methylcoumarin; C012843:cinnamaldehyde
keywords: GeLC-MS/MS; contact allergy; proteomics; sensitizer; skin