Česká společnost pro hmotnostní spektrometrii

(CEEPC/IPM/CMSC 2022 - ThS-01)
Detection of C. difficile Toxin B by biomolecule-modified chips and mass spectrometry

Josef Dvořák ^1,2 *, Petr Pompach ^3,1, Michael Volný ², Jaroslav Hrabák ⁴, Zuzana Kalaninová ^1,2, Petr Novák ^2,1

1. Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic
2. Institute of Microbiology, The Czech Academy of Science, Prague, Czech Republic
3. Institute of Biotechnology, The Czech Academy of Science, Prague, Czech Republic
4. Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic

Abstrakt

Clostridium difficile is an important human pathogen responsible for almost 224 000 hospitalized cases and 13 000 deaths annually in the USA. This anaerobic bacterium causes diarrhea and inflammation or can even cause life-threatening pseudomembranous colitis. C. difficile uses two toxins – Toxin A and B. Toxin B catalyzes RhoA glucosylation of threonine 37, therefore inactivating its GTPase activity. The inactivation of RhoA may lead to the disruption of the intestine cytoskeleton.

This study presents a novel approach of MALDI-TOF MS-based Toxin B detection using neutravidin chips prepared by ambient ion soft-landing technology for surface modification with biomolecules. The RhoA protein with a biotin tag was prepared by recombinant expression and successfully used as a substrate in enzymatic reaction with commercially available Toxin B under previously published conditions. The biotinylation allows in-situ enrichment of RhoA protein from complex samples by neutravidin chips compatible with MALDI MS. Therefore, the in-situ enriched RhoA modified by Toxin B can be detected using MALDI TOF mass spectrometry. This study’s main aim is to prepare a simple kit that allows the detection of Toxin B presence in complex samples of patients suffering C. difficile infection.

* Korespondující autor: dvorakjos@natur.cuni.cz

Poděkování:

Medical Research Foundation of the Czech Republic (NV19-05-00541) Czech Academy of Sciences (RVO61388971) Charles University Grant Agency (384021) Center of molecular structure Core Facility (CZ.1.05/1.1.00/02.0109) Czech Infrastructure for Integrative Structural Biology (LM2018127)