Direct Peptide Recognition Assay (DPRA)
direct peptides are small fragments of proteins, typically containing two or more amino acid residues, that are used to identify protein sequences. They are also the starting point for many structural characterization techniques, such as mass spectrometry.
Mass spectrometry (MS) is a powerful tool in biotechnology and pharmacology, and can be used to determine the structure of proteins, peptides and other biological molecules. Its versatility is reflected by the wide range of applications that have been developed for this technique. These include the identification and characterisation of biopolymers, such as proteins and peptides, and the separation of lipids and other macromolecules. It can also be used to analyse the metabolism of cells, for instance to study the distribution of metabolites within a cell or a tissue.
The DPRA is an in vivo test method that models the first step of the skin sensitization Adverse Outcome Pathway (AOP) by mimicking epidermal protein binding to chemical substances that are electrophilic in nature. The assay is based on the reactivity of the test substance towards synthetic peptides that contain the nucleophilic amino acid residues lysine and cysteine.
During the incubation, the test substance reacts with the peptides, resulting in a depletion of the free amino acids in the sample. This is measured by high-pressure liquid chromatography (HPLC) with UV detection. The average peptide depletion data for both cysteine and lysine are calculated, and the result is expressed as an indicator of the sensitization potential of the tested material.
To enhance the sensitivity of the assay, it can be carried out with postsource decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This technique allows for the fragmentation of precursor ions that are eluted from the ion source of the MALDI-TOF instrument. The fragment ions are then separated by the reflectron of the instrument, providing structural information for peptides with molecular masses less than 2000 Da.
In this application, the DPRA is used to evaluate the potential of essential oils to cause skin irritation. The results from the in chemico DPRA assay are compared to the known skin sensitization potential of these oils, determined by in vitro tests. The results from this study demonstrate that DPRA is an effective in vivo test to predict the skin sensitization potential of essential oils. Using this approach, the DPRA is a valuable alternative to in vivo skin sensitization testing methods and can help reduce animal testing. This research was supported by the European Union Horizon 2020 Framework Programme project BIOPharma Product Testing Munich GmbH. The EURL ECVAM is accredited to perform the DPRA test according to OECD guidance OECD 442C.