1. Characterization of β-lactamase enzyme activity in bacterial lysates using MALDI-mass spectrometry.
- Author
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Hooff GP, van Kampen JJ, Meesters RJ, van Belkum A, Goessens WH, and Luider TM
- Subjects
- Calibration, Escherichia coli enzymology, Kinetics, Penicillin G analogs & derivatives, Penicillin G chemistry, Reference Standards, Reproducibility of Results, Bacterial Proteins chemistry, Enzyme Assays methods, Escherichia coli Proteins chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization standards, beta-Lactamases chemistry
- Abstract
Plasmid-encoded β-lactamases are a major reason for antibiotic resistance in gram negative bacteria. These enzymes hydrolyze the β-lactam ring structure of certain β-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of β-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine β-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI time-of-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the β-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible β-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of β-lactamase activity in various bacterial strains by mass spectrometry.
- Published
- 2012
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