Evaluation of protein extraction protocols for MALDI-TOF Biotyper analysis of mycobacteria.

Publication date: Dec 01, 2024

Infections caused by Mycobacterium tuberculosis and nontuberculous mycobacteria represent a significant global threat and medical concern. Therefore, accurate and reliable methods must be employed to identify mycobacteria rapidly. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique that compares the cellular protein profiles of unknown isolates with reference mass spectra in a database to identify microorganisms. However, the thick and waxy lipid layer, which is rich in mycolic acids and is present in mycobacterial cells, makes protein extraction challenging. To identify the optimal protocol for correctly identifying bacilli using MALDI-TOF mass spectrometry, this study compared four different cellular protein extraction methods. Four strains of M. bovis BCG were selected as representatives of slow-growing mycobacteria, while three strains of fast-growing mycobacteria were also included: M. peregrinum, M. smegmatis, and M. farcinogenes. The extraction method that proved most effective was the extraction of inactivated cells with chloroform and methanol, which partially delipidates the cells. These cells were then extracted with formic acid, as is standard practice for protein extraction. The advantage of this method is that it allows the parallel analysis of cellular lipids and proteins from a single sample. It is therefore important to optimize mycobacterial protein extraction for MALDI-TOF MS analysis in clinical microbiology laboratories.

Concepts Keywords
Fast Bacterial Proteins
Inactivated Bacterial Proteins
Mycobacteria Bacterial Typing Techniques
Spectrometry Chloroform
Tuberculosis Chloroform
Formates
Formates
formic acid
Humans
Lipid profiling
MALDI-TOF MS
Mycobacteria identification
Mycobacterium
Mycobacterium bovis
Mycobacterium bovis BCG
Nontuberculous Mycobacteria
NTM
Protein extraction protocol
Sample preparation

Semantics

Type Source Name
disease IDO protein
disease MESH Infections
drug DRUGBANK BCG vaccine
drug DRUGBANK Formic Acid

Original Article

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