Performance comparison of three trypsin columns used in liquid chromatography

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11310%2F17%3A10368328" target="_blank" >RIV/00216208:11310/17:10368328 - isvavai.cz</a>

Výsledek na webu

<a href="https://doi.org/10.1016/j.chroma.2017.02.024" target="_blank" >https://doi.org/10.1016/j.chroma.2017.02.024</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.chroma.2017.02.024" target="_blank" >10.1016/j.chroma.2017.02.024</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Performance comparison of three trypsin columns used in liquid chromatography

Popis výsledku v původním jazyce

Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300 (A) over circle pores, packed in 2.1 x 30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using N alpha-Benzoyl-L-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37 degrees C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented.

Název v anglickém jazyce

Performance comparison of three trypsin columns used in liquid chromatography

Popis výsledku anglicky

Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300 (A) over circle pores, packed in 2.1 x 30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using N alpha-Benzoyl-L-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37 degrees C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10406 - Analytical chemistry

Projekt

<a href="/cs/project/GA16-05942S" target="_blank" >GA16-05942S: Gradientová chromatofokusace a izoelektrická fokusace - nástroje pro separaci proteinů</a><br>

Návaznosti

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

Rok uplatnění

2017

Kód důvěrnosti údajů

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

Název periodika

Journal of Chromatography A

ISSN

0021-9673

e-ISSN

—

Svazek periodika

1490

Číslo periodika v rámci svazku

March

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

7

Strana od-do

126-132

Kód UT WoS článku

000397375600015

EID výsledku v databázi Scopus

2-s2.0-85013067919