BiotinSwitch Profiling of skin sensitizers

ABSTRACT: Allergic contact dermatitis (ACD) is one of the most common form of allergies. It affects about 20% of the population. It is a T-cell mediated hypersensitivity against contact allergens, haptens. The haptens are small electrophilic molecules that can covalently bind to nucleophilic peptides of the skin proteins. This process is called haptenation and it is the first key molecular event in the adverse outcome pathway. Due to the ban on animal testing in EU, alternative testing methods are needed to accurately access the sensitisation potential of compounds. Currently there are no efficient strategies to discover the target proteins or the pathways involved in haptenation. We report activity-based profiling (ABP) screening to identify the proteins involved in haptenation during ACD. Our approach is applicable to both haptens and pre-/pro-haptens compounds. Using this method, we have identified proteins that are potentially targeted by 17 electrophilic compounds with three different sensitizing potentials: strong, moderate and weak/non-sensitizer. The compound treated cells are harvested, lysed and proteins extracted. The soluble fraction is biotinylated with cysteine reactive biotin probe. The biotinylated proteins are enriched using streptavidin coated magnetic beads. The proteins are digested, desalted and subjected to high pH fractionation into 4 fractions. The fractionated peptides are injected into Thermo Fischer Scientific Orbitrap HF-X mass analyser coupled with EasynLC 1200. The mass spectra is acquired in data dependent acquisition mode. The acquired spectra are searched in Proteome Discoverer. The sensitizing potential of a compound can be evaluated by the number of protein targets that are identified using biotin switch assay. The common proteins that are repeatedly targeted by different sensitizers are identified and the corresponding enriched pathways and organelles were identified.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Radoslaw M. Sobota 

PROVIDER: PXD033313 | JPOST Repository | Thu Apr 20 00:00:00 BST 2023

REPOSITORIES: jPOST