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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61989592%3A15310%2F20%3A73603593" target="_blank" >RIV/61989592:15310/20:73603593 - isvavai.cz</a>

Výsledek na webu

<a href="https://onlinelibrary.wiley.com/doi/full/10.1002/jms.4444" target="_blank" >https://onlinelibrary.wiley.com/doi/full/10.1002/jms.4444</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1002/jms.4444" target="_blank" >10.1002/jms.4444</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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 mass spectrometry (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. Matrix‐assisted laser desorption/ionization (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 with 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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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 mass spectrometry (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. Matrix‐assisted laser desorption/ionization (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 with 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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/EF16_019%2F0000827" target="_blank" >EF16_019/0000827: Rostliny jako prostředek udržitelného globálního rozvoje</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2020

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

JOURNAL OF MASS SPECTROMETRY

ISSN

1076-5174

e-ISSN

—

Svazek periodika

55

Číslo periodika v rámci svazku

7

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

8

Strana od-do

"e4444-1"-"e4444-8"

Kód UT WoS článku

000552073200011

EID výsledku v databázi Scopus

2-s2.0-85078809133