1. High throughput detection of deamidation using S-(5'-adenosyl)-l-homocysteine hydrolase and a fluorogenic reagent.
- Author
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Murphy BM, Ozumerzifon TJ, Henry CS, and Manning MC
- Subjects
- Amides metabolism, Amino Acid Sequence, Asparagine chemistry, Asparagine metabolism, Enzyme Assays economics, Enzyme Assays instrumentation, Enzyme Assays methods, Glucagon chemistry, High-Throughput Screening Assays economics, High-Throughput Screening Assays instrumentation, Hydrogen-Ion Concentration, Proteolysis, Sensitivity and Specificity, Sodium Chloride chemistry, Adenosylhomocysteinase chemistry, Fluorescent Dyes chemistry, High-Throughput Screening Assays methods
- Abstract
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., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
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