Mixed-mode column allows simple direct coupling with immobilized enzymatic reactor for on-line protein digestion

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11310%2F23%3A10470988" target="_blank" >RIV/00216208:11310/23:10470988 - isvavai.cz</a>

Výsledek na webu

<a href="https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=wCDqvHyMP1" target="_blank" >https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=wCDqvHyMP1</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.jchromb.2023.123866" target="_blank" >10.1016/j.jchromb.2023.123866</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Mixed-mode column allows simple direct coupling with immobilized enzymatic reactor for on-line protein digestion

Popis výsledku v původním jazyce

Liquid chromatography coupled with mass spectrometry is widely used in the field of proteomic analysis after off-line protein digestion. On-line digestion with chromatographic column connected in a series with immobilized enzymatic reactor is not often used approach. In this work we investigated the impact of chromatographic conditions on the protein digestion efficiency. The investigation of trypsin reactor activity was performed by online digestion of N-alpha-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA), followed by separation of the digests on the mixed-mode column. Two trypsin column reactors with the different trypsin coverage on the bridged ethylene hybrid particles were evaluated. To ensure optimal trypsin activity, the separation temperature was set at 37.0 degrees C and the pH of the mobile phase buffer was maintained at 8.5. The on-line digestion itself ongoing during the initial state of gradient was carried out at a low flow rate using a mobile phase that was free of organic modifiers. Proteins such as cytochrome C, enolase, and myoglobin were successfully digested on-line without prior reduction or alkylation, and the resulting peptides were separated using a mixed-mode column. Additionally, proteins that contain multiple cysteines, such as alpha-lactalbumin, albumin, beta-lactoglobulin A, and conalbumin, were also successfully digested online (after reduction and alkylation). Moreover, trypsin immobilized enzymatic reactors were utilized for over 300 injections without any noticeable loss of digestion activity.

Název v anglickém jazyce

Mixed-mode column allows simple direct coupling with immobilized enzymatic reactor for on-line protein digestion

Popis výsledku anglicky

Liquid chromatography coupled with mass spectrometry is widely used in the field of proteomic analysis after off-line protein digestion. On-line digestion with chromatographic column connected in a series with immobilized enzymatic reactor is not often used approach. In this work we investigated the impact of chromatographic conditions on the protein digestion efficiency. The investigation of trypsin reactor activity was performed by online digestion of N-alpha-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA), followed by separation of the digests on the mixed-mode column. Two trypsin column reactors with the different trypsin coverage on the bridged ethylene hybrid particles were evaluated. To ensure optimal trypsin activity, the separation temperature was set at 37.0 degrees C and the pH of the mobile phase buffer was maintained at 8.5. The on-line digestion itself ongoing during the initial state of gradient was carried out at a low flow rate using a mobile phase that was free of organic modifiers. Proteins such as cytochrome C, enolase, and myoglobin were successfully digested on-line without prior reduction or alkylation, and the resulting peptides were separated using a mixed-mode column. Additionally, proteins that contain multiple cysteines, such as alpha-lactalbumin, albumin, beta-lactoglobulin A, and conalbumin, were also successfully digested online (after reduction and alkylation). Moreover, trypsin immobilized enzymatic reactors were utilized for over 300 injections without any noticeable loss of digestion activity.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10406 - Analytical chemistry

Projekt

<a href="/cs/project/GA20-19655S" target="_blank" >GA20-19655S: Strategie pro on-line štěpení proteinů s následnou separací v mix-mode chromatografii</a><br>

Návaznosti

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

Rok uplatnění

2023

Kód důvěrnosti údajů

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

Název periodika

Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

ISSN

1570-0232

e-ISSN

1873-376X

Svazek periodika

1228

Číslo periodika v rámci svazku

August

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

8

Strana od-do

123866

Kód UT WoS článku

001077282500001

EID výsledku v databázi Scopus

2-s2.0-85168998793