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

Result code in 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>

Result on the web

<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>

Result language

angličtina

Original language name

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

Original language description

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.

Czech name

—

Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10406 - Analytical chemistry

Project

<a href="/en/project/GA20-19655S" target="_blank" >GA20-19655S: Strategy for on-line protein digestion followed by separation in mixed-mode chromatography</a><br>

Continuities

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

Publication year

2023

Confidentiality

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

Name of the periodical

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

ISSN

1570-0232

e-ISSN

1873-376X

Volume of the periodical

1228

Issue of the periodical within the volume

August

Country of publishing house

NL - THE KINGDOM OF THE NETHERLANDS

Number of pages

8

Pages from-to

123866

UT code for WoS article

001077282500001

EID of the result in the Scopus database

2-s2.0-85168998793