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Mass spectrometry of peptides and proteins using digestion by a grape cysteine protease at pH 3.

08:00 EDT 11th October 2019 | BioPortfolio

Summary of "Mass spectrometry of peptides and proteins using digestion by a grape cysteine protease at pH 3."

Cysteine protease from grapevine (Vitis vinifera) belongs to those resistant proteins, which survive the process of vinification and can therefore be detected as wine components. Its amino acid sequence shows a homology to other members of the papain family but the enzyme has only partially been explored so far. In order to get more biochemical information with the help of MS, wine proteins were collected by ultrafiltration and separated by gel permeation chromatography. The purified enzyme surprisingly displayed a high molecular mass value of around 200 kDa, indicating a possible oligomeric status and aggregation, as it entered only negligibly the separating 10% gel during polyacrylamide gel electrophoresis. The isoelectric point (pI) value of 3.6 was determined by chromatofocusing. MALDI-MS was employed to evaluate the cleavage specificity and usefulness of the isolated cysteine protease in protein and peptide research. A potential applicability could be anticipated from the efficient digestion performance in volatile ammonium formate buffers at pH 3. Common peptides were digested and the resulting products analyzed by MS/MS sequencing. Then, mixtures of protein standards and extracted barley nuclear proteins were processed in the same way. Grape cysteine protease is nonspecific but shows a certain preference for Arg, Lys and also Leu residues. Compared to papain it seems not to require fully the presence of a large hydrophobic residue adjacent to that at the cleavage site. The enzyme is suitable for protein research as it produces peptides of a reasonable length in acidic pH.

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This article was published in the following journal.

Name: Journal of mass spectrometry : JMS
ISSN: 1096-9888
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Medical and Biotech [MESH] Definitions

Analysis of PEPTIDES that are generated from the digestion or fragmentation of a protein or mixture of PROTEINS, by ELECTROPHORESIS; CHROMATOGRAPHY; or MASS SPECTROMETRY. The resulting peptide fingerprints are analyzed for a variety of purposes including the identification of the proteins in a sample, GENETIC POLYMORPHISMS, patterns of gene expression, and patterns diagnostic for diseases.

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