Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61989592%3A15310%2F11%3A33118642" target="_blank" >RIV/61989592:15310/11:33118642 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/60162694:G44__/11:00002600

Výsledek na webu

<a href="http://dx.doi.org/10.1016/j.jprot.2011.02.011" target="_blank" >http://dx.doi.org/10.1016/j.jprot.2011.02.011</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.jprot.2011.02.011" target="_blank" >10.1016/j.jprot.2011.02.011</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests

Popis výsledku v původním jazyce

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligandbinding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards an

Název v anglickém jazyce

Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests

Popis výsledku anglicky

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligandbinding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards an

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

CE - Biochemie

OECD FORD obor

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Projekt

<a href="/cs/project/ED0007%2F01%2F01" target="_blank" >ED0007/01/01: Centrum regionu Haná pro biotechnologický a zemědělský výzkum</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>Z - Vyzkumny zamer (s odkazem do CEZ)

Rok uplatnění

2011

Kód důvěrnosti údajů

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

Název periodika

Journal of Proteomics

ISSN

1874-3919

e-ISSN

—

Svazek periodika

74

Číslo periodika v rámci svazku

7

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

10

Strana od-do

948-957

Kód UT WoS článku

000292440400005

EID výsledku v databázi Scopus

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