Tue. Feb 27th, 2024

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry is a powerful analytical technique that allows the measurement of the molecular weight of direct peptides and proteins in a complex sample. It is also capable of rapidly profiling the proteome from whole cells and small tissue samples. It requires minimal sample preparation, and its fast evaporation method has been optimized for use in the DPRA to improve reproducibility and quantitation. This article reviews MALDI-based methods for peptide and protein identification, the structural characterization of proteins, as well as the identification of posttranslational modifications. It is argued that MALDI has potential for further advancements to allow a greater understanding of the structural and functional organization of biological molecules.

A key step in determining the skin sensitizing potential of chemicals is to evaluate their ability to bind to nucleophilic centers in skin proteins. The direct peptide reactivity assay (DPRA) contributes to this evaluation by quantifying the reactivity of chemicals towards two model peptides. Based on their reaction with these peptides, chemicals can be classified into four reactivity categories and discriminated between sensitisers and non-sensitisers.

In the DPRA, the test substance is incubated with synthetic peptides containing either cysteine or lysine and their content is determined by HPLC using UV detection. The reactivity of the test substance is determined by calculating the percentage of the peptides that are depleted after incubation with the chemical. This peptide depletion is then compared with that of a negative control to determine the reactivity of the test substance. The kinetic direct peptide reactivity assay is an extension of this test that allows the reactivity of the test substance to be evaluated by assessing the reaction kinetics.

The kDPRA was developed to overcome some limitations of the original DPRA. It is a simple, easy to perform test that can be performed in parallel, and which is suitable for use by laboratories of any size. It was evaluated for transferability, proficiency, within-laboratory reproducibility, and predictive ability based on Good Laboratory Practices (GLP) principles at three different laboratories. Results indicate that kDPRA is an accurate and reliable method for classifying chemicals into the skin sensitization potency category and that it can provide a useful contribution to evaluating the reactivity of substances towards skin proteins. Further work is needed to develop and validate the kDPRA for the assessment of respiratory sensitizers. Further, it is important to establish a standardized procedure for preparing samples and calibrating the kDPRA. This will help to ensure the quality of kDPRA data and enable the development of automated testing systems that will further increase the utility of this method. 2011 Wiley Periodicals, Inc. All rights reserved. Reproduction of this article in any form without written permission from the publisher is prohibited. This material is available through a subscription to Wiley InterScience. Request a free trial or purchase online at our store. Click here for more information.

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