A peptide is an amino acid that forms part of a protein. These molecules are used as markers for the protein’s structure and function, and can be used to identify amino acids within a sample. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a well-established analytical technique for obtaining structural information from biological samples including cells and tissues. MALDI-TOF MS is also a good choice for analyzing large volumes of small biomolecules, such as proteins and direct peptides because it provides high mass resolution, enabling identification and quantification at a single-molecule level.
The direct peptide reactivity assay (DPRA) contributes to the assessment of the skin sensitization potential of chemicals, and is one of three test methods that can replace animal testing for determining potency. The DPRA is based on the concept that chemical allergens bind to proteins and that these interactions with proteins can initiate a hypersensitivity reaction in humans. It is therefore a useful screen for chemical ingredients with the potential to cause allergic reactions of the skin and respiratory tract.
In the DPRA, known skin and respiratory sensitizers are reacted with synthetic cysteine and lysine-containing peptides for 24 hours. The amount of peptide depleted by the chemical is determined using HPLC/UV. Known respiratory allergens are shown to preferentially react with the lysine peptide, whereas most skin sensitizers react with cysteine.
The DPRA is modified by incorporating the use of monobromobimane in the test procedure. This compound is added to the peptide solution to form a fluorescent complex with any free cysteine residues of the model peptide. The amount of fluorescently eluted peptide reflects the quantity of free cysteine in the sample. The kinetic direct peptide reactivity assay, or kDPRA, is a variation of the DPRA that measures the reactivity of a sample at different incubation times with the synthetic peptides.
All the essential oils tested were soluble in acetonitrile. A 100 mM stock solution of each oil was prepared fresh, immediately prior to analysis. The stocks were diluted with acetonitrile to the correct concentration for each assay. One blank sample and a control of each essential oil were also prepared. The essential oils were tested for their ability to absorb at 220 nm and for co-elution with the peptides. The results were compared to the published data of Gerberick et al., 2007.