Dataset Information

A method for producing heat-stable and pure trypsin

ABSTRACT: In this study we evaluated several different strategies for reductive methylation and purification of porcine trypsin in order to write a protocol that can be followed by proteomics laboratories.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Equus Caballus (horse) Homo Sapiens (human) Bos Taurus (bovine)

SUBMITTER: Søren Heissel

LAB HEAD: Peter Højrup

PROVIDER: PXD008193 | Pride | 2019-06-06

REPOSITORIES: Pride

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			Action	DRS
	2percent_Specific_SEARCH.zip	Other
	2percent_Unspecific_SEARCH.zip	Other
	4percent_Specific_SEARCH.zip	Other
	4percent_Unspecific_SEARCH.zip	Other
	Promega1.raw	Raw

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Publications

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

Heissel Søren S Frederiksen Sigurd J SJ Bunkenborg Jakob J Højrup Peter P

PloS one 20190627 6

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 ...[more]

PMID: 31246970

Similar Datasets

Project description:1. Isoionic chemical modification of amino groups of trypsin (EC 3.4.21.4) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The alpha-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of beta-trypsin retained enzymic activity at values comparable with those of native enzyme tested with alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of alpha-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated beta-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus beta-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated alpha-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the alpha-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p'-guanidinobenzoate. 5. It was concluded that acetamidination of beta-trypsin is a convenient method for preparing a well-defined stable and soluble trypsin derivative without appreciable change in its physical properties.