Mass Spectrometry-Based Solid Phase Peptide Reaction Assay for Detecting Allergenicity Using an Immobilized Peptide-Conjugating Photo-Cleavable Linker.

The biological process of skin sensitization depends on the ability of a sensitizer to modify endogenous proteins. A direct peptide reactivity assay (DPRA), based on the biological process of skin sensitization, was developed as an alternative to controversial animal experiments. Although DPRA has been endorsed by industries and is internationally accepted as promising, it has several drawbacks, such as incompatibility with hydrophobic chemicals, inability to perform detailed reaction analysis, and ability to evaluate only single components. Here, we demonstrated that sensitizers and peptide adducts can be easily identified using a mass spectrometry-based solid-phase peptide reaction assay (M-SPRA). We synthesized peptides with a photo-cleavable linker immobilized on resins. We showed the potential of M-SPRA in predicting skin sensitization by measuring the peptide adducts that were selectively eluted from the resin after cleaving the linker post-reaction. M-SPRA provides more detailed information regarding chemical reactivity and accurate assessment of test samples, including mixtures. M-SPRA may be helpful for understanding the binding mechanism of sensitizers (toxicology), which may assist in further refining reactivity assays and aiding in the interpretation of reactivity data.

Development of a chromophore-solid phase peptide reaction assay (C-SPRA) for assessing skin sensitization in vitro.

We developed an in vitro chromophore-solid phase peptide reaction assay (C-SPRA) using microbead-immobilized peptides and chromophores. Peptide-resins (microbeads) reacted with 14 representative chemicals to demonstrate the test's capacity to predict skin sensitization. C-SPRA enables accurate and high-throughput assessments of various chemicals, including poorly water-soluble sensitizers that are regarded as weakly potent by other methods.