Příprava afinitních povrchů pro hmotnostní spektrometrii

• Main Author: Kalaninová, Zuzana
• Other Authors: Pompach, Petr
• Format: Dissertation
• Language: Czech
• Published: 2021
• Online Access: http://www.nusl.cz/ntk/nusl-449070

One of the most potent toxic substances of biological origin known are botulinum toxins - nerve toxins produced by strains of Clostridium bacteria. They affect neuro-muscular connections where they cleave proteins of the SNARE complex, block neurotransmitter release, and thus stop signal transmission. This leads to flaccid paralysis, the main symptom of a disease called botulism. Due to their extremely low lethal dose and high toxicity, BoNTs take a place on the list of agents of bioterrorism. Despite numerous methods available to this date, the mouse bioassay remains the gold standard for the detection of BoNTs. Regardless of the ethical aspects of these experiments, the time they require is also inconvenient. In this work, a unique combination of protein surfaces and detection of active BoNTs through their endopeptidase activity and mass spectrometry, EndoPep-MS, is introduced. Functionalized surfaces were prepared by ambient ion soft landing - a technique utilizing a beam of hyperthermal ions (energy range 1-100 eV) as a tool for controlled modification. Two approaches to the detection of botulinum toxin A1 were applied. One of them, based on avidin- biotin interaction, used a neutravidin-modified surface for enhancement of biotinylated peptides (substrate peptide for BoNT/A and peptide products...