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

Søren Heissel, Sigurd J Frederiksen, Jakob Bunkenborg, Peter Højrup

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Abstract

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.

Original languageEnglish
Article numbere0218374
JournalPLOS ONE
Volume14
Issue number6
Number of pages16
ISSN1932-6203
DOIs
Publication statusPublished - 27. Jun 2019

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High temperature applications
proteomics
Trypsin
trypsin
Purification
Stabilization
Methylation
temperature
methylation
Peptide Hydrolases
proteinases
digestion
Workflow
Chemical modification
purity
Sepharose
agarose
Proteomics
Enzymes
enzymes

Cite this

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abstract = "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.",
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Enhanced trypsin on a budget : Stabilization, purification and high-temperature application of inexpensive commercial trypsin for proteomics applications. / Heissel, Søren; Frederiksen, Sigurd J; Bunkenborg, Jakob; Højrup, Peter.

In: PLOS ONE, Vol. 14, No. 6, e0218374, 27.06.2019.

Research output: Contribution to journalJournal articleResearchpeer-review

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T2 - Stabilization, purification and high-temperature application of inexpensive commercial trypsin for proteomics applications

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AU - Bunkenborg, Jakob

AU - Højrup, Peter

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AB - 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.

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