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Substrate Profiling

Substrate selectivity or specificity is the key to successful development of biocatalysts. As a natural catalyst, enzyme inherently presents promiscuity over similar molecules. Therefore, understanding substrate specificity of a particular enzyme holds an important place in Biocatalysis development. Substrate profiling is a process to set rapid assays for purified proteins or complex biological mixtures with different substrates to study substrate specificity of those biocatalysts. It facilitates new groupings of enzymes based on the chemical transformations that they catalyze and the small molecules with which they interact rather than on sequence or structural properties. It is powerful tool of biochemical proteomics, particularly since many sequence and structurally related proteins can perform largely different chemistry.

Substrate profiling is applied to various biocatalysts. Using hydrolases as an example, commercially available phosphorylated compounds can be used for phosphatase screen based on detection of released Pi with the highly sensitive Malachite Green reagent. Recently, synthetic peptide libraries are constructed to study the substrate specificity of certain biocatalysts, such as kinases and proteases. This procedure is known as multiplex substrate profiling (MSP). In these cases, purified enzymes or complex biological mixtures can be added to a peptide library, and the resulting modified peptide products are directly monitored throughout the reaction by peptide sequencing via liquid chromatography−tandem mass spectrometry (LC−MS/MS).

Creative Enzymes offers the service of substrate profiling, aimed at evaluation of substrate specification of a known bio-catalysis reaction. Enabled by our best knowledge of bioinformatic technique, we offer professional one-stop services:


  1. Kuznetsova, E., Proudfoot, M., Sanders, S.A., Reinking, J., Savchenko, A., Arrowsmith, C.H., Edwards, A.M., Yakunin, A.F. (2005) Enzyme genomics: Application of general enzymatic screens to discover new enzymes. FEMS Microbiology Reviews. 29: 263-279.
  2. Meyer, N.O., O'Donoghue, A.J., Schulze-Gahmen, U., Ravalin, M., Moss, S.M., Winter, M.B., Knudsen, G.M., Craik, C.S. (2017) Multiplex Substrate Profiling by Mass Spectrometry for Kinases as a Method for Revealing Quantitative Substrate Motifs. Analytical Chemistry. 89: 4550-4558.

An example of MSP-MS assay for kinaseFigure 1. An example of MSP-MS assay for kinase
(Analytical Chemistry, 2017)

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