High throughput detection of deamidation using S-(5′-adenosyl)-l-homocysteine hydrolase and a fluorogenic reagent


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Abstract

Graphical abstractHighlightsThe fluorogenic assay using TFP4 was used to monitor the time course of deamidation of glucagon at pH 10.The method indicates that NaCl can inhibit deamidation of glucagon under basic pH conditions.The sensitivity of this assay is comparable to the commercially available IsoQuant kit.Deamidation of asparagine (Asn) residues is one of the most common chemical degradation pathways observed in proteins. This reaction must be understood and controlled in therapeutic drug candidates, as chemical changes can affect their efficacy and safety. The analytical tools available for detection of deamidation reaction products, such as isoaspartic acid residues, are either chromatographic or electrophoretic, and require MS detection for absolute identification of peaks. High-throughput measurement of protein degradation has typically been limited to probing the target's physical state using spectroscopic techniques. Here, we describe a high throughput assay for isoaspartate residues using fluorescent detection in a microtiter plate format. The method allows for fast detection of protein deamidation in a cost-efficient manner. The method can be employed even if the target peptide or protein contains free Cys residues. The technique appears to be selective, linear, and accurate.

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