The Use of Direct Peptides in Skin Sensitisation Assays
direct peptides are fragments of proteins that can be used to identify protein sequence. They are usually produced by a proteolytic enzyme and injected into the sample for detection with mass spectrometry. Peptide fragmentation allows for high resolution analysis of biological samples with minimal protein loss. In contrast to conventional peptide sequencing methods, which are based on gel electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) allows direct peptide profiling and identification of peptides with molecular masses up to 2000 Da. MALDI-TOF MS employs a tandem double-focusing magnetic-orthogonal acceleration time-of-flight mass spectrometer. This device retards and reverses the ion flight paths of ions with different kinetic energies, thereby increasing the mass resolution of the instrument.
The in chemico Direct Peptide Reactivity Assay (DPRA) is one of three test methods that contribute to the assessment of skin sensitisation potential of chemicals, pharmaceuticals and cosmetics/personal care products (KeratinoSensTM and h-CLAT). This chemical screening assay, also known as a Molecular Initiating Event (MIE), addresses epidermal protein binding to artificial peptides by measuring the depletion of cysteine or lysine from the reaction mixture following incubation for 24 hours. The result is a score of 0–3 which classifies the chemicals as having minimal, low, medium or high reactivity with these synthetic peptides, and thus potentially, with epidermal proteins.
DPRA is a chemistry-based method that incubates the test substance with a solution of two nucleophile-containing peptides, cysteine and lysine, for 24 hours at room temperature. The rate of peptide depletion from the reaction mixture is then measured by high-pressure liquid chromatography (HPLC) using UV detection. The resulting data is then assessed by a software program that assigns the test substance a score of 0–3. The DPRA result can then be compared with the corresponding DPRA scores of the reference list of known skin sensitizers to determine the likely sensitivity of the test substance.
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The DPRA is an alternative to animal testing and has been independently peer reviewed, endorsed and approved by EURL ECVAM as a Validated Test Method for OECD Guidance OECD 442C. The test has also been endorsed by the European Commission as a Test Method of Alternative Approaches (TMB59).
Our laboratories have the capabilities to perform in-house MALDI-TOF mass spectrometric analysis and immunological studies for both research and commercial purposes. We are proud to provide high-quality service, expert advice and competitive pricing. We look forward to assisting you with your future projects.