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Disulfide Bond Analysis

Disulfide bonds are a common posttranslational modification in proteins, which are formed between the sulfur atoms of two cysteine (Cys) residues during biosynthesis of proteins. Due to is wide presence, analysis of disulfide bonds are often critical to enzyme discovery and identification.

Compared to other proteins, disulfide bonds are especially critical for enzymes in proper folding, and they have both structural and functional roles. For example, non-native disulfide bonds can be engineered into enzymes to increase its stability. Also, some disulfide bonds, known as allosteric disulfides, are responsible for effective biological functions of enzymes, and the cleavage of such bonds would lead to a change in the protein activity. Therefore, disulfide bond analysis provides key information to research pertaining to protein stability, structure-function relationships, and any disulfide-mediated isoforms of enzymes.

Disulfide bonds can be analyzed mainly by three classes of techniques: i) Crystallographic techniques producing high-resolution three-dimensional structures. ii) Algorithmic techniques based on sequence data. iii) Techniques combing experimental and algorithmic methods to analyze a mixture of digested peptides. A variety of analytical techniques are involved in a typical study, such as NMR spectroscopy, X-ray crystallography, Edman degradation, diagonal paper electrophoresis, and liquid chromatography (LC) coupled with mass spectrometry (MS). Comparing to limitations in other methods, such as high purity sample requirements and low throughput, bottom-up MS is the most widely used method for disulfide bond analysis. In general, this bottom-up approach involves digestion of large biomolecules into small pieces that are easy to analyze, with the help of ionization techniques, complementary fragmentation techniques and LC system for separation.  

Creative Enzymes provides highly sensitive HPLC-MS/MS platform that can analyze disulfide bonds in multiple samples from various sources. In addition, we develop advanced machine-learning strategies based on sequence-based information. Furthermore, we provide services regarding to disulfide artifacts verification and disulfide engineering. Our featured services include:

Reference

Lakbub, J.C., Shipman, J.T., Desaire, H. (2018) Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins. Analytical and Bioanalytical Chemistry. 410(10): 2467-2484.

LC-MS/MS method aiming for disulfide bond analysis. Figure 1 A typical disulfide bond (DSB) analysis workflow.
(Analytical and Bioanalytical Chemistry 2018)



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