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Enhanced trypsin on a budget: Stabilization, purification and high-temperature application of inexpensive commercial trypsin for proteomics applications

Trypsin is by far the most commonly used protease in proteomics. Even though the amount of protease used in each experiment is very small, digestion of large amounts of protein prior to enrichment can be rather costly. The price of commercial trypsin is highly dependent on the quality of the enzyme,...

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Detalles Bibliográficos
Autores principales: Heissel, Søren, Frederiksen, Sigurd J., Bunkenborg, Jakob, Højrup, Peter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597055/
https://www.ncbi.nlm.nih.gov/pubmed/31246970
http://dx.doi.org/10.1371/journal.pone.0218374
Descripción
Sumario:Trypsin is by far the most commonly used protease in proteomics. Even though the amount of protease used in each experiment is very small, digestion of large amounts of protein prior to enrichment can be rather costly. The price of commercial trypsin is highly dependent on the quality of the enzyme, which is determined by its purity, activity, and chemical modifications. In this study we evaluated several strategies for improving the quality of crude trypsin by reductive methylation and affinity purification. We present a protocol applicable to most proteomics laboratories for obtaining a highly stable and pure trypsin preparation using reductive methylation and purification by benzamidine-sepharose. The entire workflow can be performed within a day and yields ~4 mg per batch but is completely scalable. The methylated product was benchmarked against sequencing grade trypsin from Promega and they were found to be comparable for one hour digestions at elevated temperatures, where residual chymotryptic activity was found to be negligible.