Direct Peptides and MALDI-TOF MS

Direct peptides are a class of amino acids found in proteins that act as nucleophilic centres to react with and attack the polar groups of other molecules. They are the building blocks of amino acid sequences, making them the most common components of peptides and proteins in living systems and forming the basis for the structure of biologically important compounds such as hormones and enzymes. The peptides themselves are a class of biomolecules that can be directly identified, measured and analysed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) techniques. This rapid, highly sensitive technique is well suited to the characterisation of biomolecules in complex samples without prior fractionation, isolation or other sample preparation steps. This article presents a series of examples where MALDI-TOF MS is used to identify, measure and analyse peptides and proteins in different environments, including living cells, tissues and cellular extracts. It also outlines a range of recent enhancements in MALDI-TOF MS that enable the identification and quantification of biomolecules, particularly peptides.

MALDI-TOF MS is a technique that is widely used in the pharmaceutical industry to identify and measure peptides and proteins in biological tissues, cell lysates and other complex mixtures. The technique is particularly suited to the characterisation of proteomics samples that contain large numbers of protein fragments with a wide spectrum of molecular weights. The MALDI-TOF MS method is also able to identify and quantify post-translational modifications that alter protein function.

DPRA is an in-vitro test for skin sensitisation that models one of the key events of the skin sensitisation Adverse Outcome Pathway (AOP). The DPRA chemistry based assay assesses the ability of chemicals to interact with and covalently bind to the cysteine or lysine residues of two synthetic peptides. Following 24 hours of incubation, the peptides are depleted of their free cysteine and lysine by the test chemical and the relative proportion of non-depleted peptide is measured. This is then used to categorise the test chemical as having low, medium or high reactivity which provides an indication of the likelihood that the chemical may cause skin sensitisation.

This test is approved as an alternative to animal testing by the OECD and has been adopted as a European Union GHS Category 1A Alternative Test Method. The modified Spectro-DPRA is the most advanced version of this assay that has been developed to overcome limitations in the original DPRA and provides a faster, more accurate and predictive result. It is available as a high-throughput, LC/MS assay to meet the needs of high volume laboratories.

Spectro-DPRA has been extensively pre-validated in accordance with EURL ECVAM Good Laboratory Practice (GLP) principles in three laboratories. This validation confirmed excellent transferability, proficiency, between- and within-laboratory reproducibility and predictive ability to evaluate the reactivity of chemical substances towards two model peptides. This makes Spectro-DPRA an extremely versatile tool to support the assessment of human skin reactivity for the hazard prediction of chemicals that are not tested with the OECD DPRA test and other in vitro methods.

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