Protein Extraction From <i>Mycobacterium</i> spp. for Identification by MALDI‐TOF Mass Spectrometry

The identification of mycobacteria remains challenging due to the high species diversity within the genus and the lack of a molecular method able to identify them in a single assay. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used as a rapid and promising alternative for bacterial identification; however, its reliability is highly dependent on effective protein extraction. This article describes a protocol specifically developed for the identification and species-level discrimination of Mycobacterium spp. that integrates thermal inactivation, multiple biomass washes, mechanical disruption with zirconia beads, protein extraction using formic acid and acetonitrile, quality control, and troubleshooting. This procedure is compatible with Bruker MALDI Biotyper systems and is designed to meet biosafety level (BSL)-2 and BSL-3 laboratory requirements, ensuring operator safety and preventing aerosol generation. Emphasis is placed on standardizing critical steps, reproducibility, and safe handling of pathogenic strains. This protocol can be applied in both diagnostic and research laboratories, offering a reliable and standardized approach to preparing mycobacterial samples for analysis by MALDI-TOF MS. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Protein extraction from Mycobacterium spp. cultured on solid media for identification by MALDI-TOF mass spectrometry Support Protocol: Preparation and quality control of mycobacterial cultures for MALDI-TOF MS analysis.