MALDI-TOF in Microbiology

Until recently, clinical diagnostic laboratories predominantly relied on conventional phenotypic ways of diagnosing infections, and sometimes on gene sequencing techniques. The latest advancements in technology include matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry which has entered quotidian microbiological practice.

MALDI-TOF mass spectrometry generates specific mass spectral fingerprints, which can be seen as unique signatures of microorganisms that may aid in their accurate identification to the genus and species levels – with vast potential to be employed for strain typing.

Bacterial Identification

Several approaches for utilizing MALDI-TOF mass spectrometry are now being used in microbiological diagnostic laboratories. One approach compares results to databases of commercially available mass spectrometry signatures to identify the sample bacteria, and a second uses a proteome database to identify biomarker masses in the bacteria from sequenced genomes. The second bioinformatics approach allows for variation differences in culture growth and sample treatment conditions, whilst first is particularly useful in routine laboratory methods, such as diagnostics, and can differentiate between species and subspecies.

Given its accuracy, the technology can also be directly applied to various clinical samples, most notably blood, cerebrospinal fluid, urine, pleural fluid, and peritoneal liquid. The major restraint is the amount of bacteria present in the samples, due to the detection limit of current MALDI-TOF protocols. To bypass this drawback, large volumes are usually required for blood and urine samples, as well as using cultures as an additional enrichment for blood.

Results observed for bacterial identification with MALDI-TOF mass spectrometry using either of the two aforementioned approaches, the diagnostic yield and accuracy highly depends on the bacterial taxonomy and the quality of used databases.

One of the principal advantages of employing MALDI-TOF technology for identifying bacteria is the rapid availability of results, which are typically ready in less than an hour. Moreover, MALDI-TOF mass spectrometry enables precise identification of a large diversity of bacteria that have scarce phenotypic traits and that necessitated 16S rRNA gene sequencing before the MALDI-TOF era.

Fungal Identification

MALDI-TOF mass spectrometry was quickly and successfully attuned for the identification of fungi. For now, this method is chiefly used for the routine yeast identification, while further development is needed (most notably in sample preparation protocols and database libraries) to make use of this identification approach for other groups of fungi (such as dermatophytes and filamentous fungi).

Akin to the situation with bacteria, misidentification or non-identification of fungal genera and species by MALDI-TOF mass spectrometry is essentially due to mistakes, absences or incomplete reference spectra in databases. The drawback is that reference spectra currently included in databases of commercially accessible MALDI-TOF mass spectrometry systems are incomplete.

Furthermore, the spectral signal pertinent to filamentous fungi depends on fungal phenotype – which includes basidiospore, fruiting body, surface mycelium and substrate mycelium. In addition, when grown on agar plate, vegetative mycelium shows manifold zones that correspond to distinct ages or stages of development. This can result in misidentification and so variable results from the same sample, therefore it is vital that databases include a thorough database of multiple MS fingerprints from different developmental stages of filamentous fungi to ensure correct identification.

Accuracy, Time and Cost effectiveness

When compared with conventional methods for microbial identification, MALDI-TOF mass spectrometry in a majority of cases confers a substantial gain of both engineer/technician working time (preparing samples) and turnaround time (obtaining results with automated analytical procedure).

However, the purchase of MALDI-TOF mass spectrometry instrument is undoubtedly one of the most expensive capital investments for the clinical microbiology laboratory. This means adequate cost justifications and cost analyzes should be pursued, which should include obligatory quality control steps.


MALDI-TOF MS is used in a variety of industries, this includes the biopharmaceutical, organic chemistry, metabolomics, and genomics, as well clinical and diagnostic and treatment applications. In organic chemistry applications MALDI-TOF is used to analyze nucleic acid, protein and polymer masses, as well as identify complex mixtures of oligonucleotides and small proteins, supplying biochemical and chemical researchers with useful information. MALDI-TOF MS plays a crucial role in the advancement of rapid patient diagnosis and improved health outcomes. A prime example of this is its use in routine classification of microorganisms in patient samples for clinical microbiology.


In conclusion, MALDI-TOF mass spectrometry is a fascinating novel technology for microbial identification that is rapid, efficient, cost-effective and simple to use. Soon this instrument will be pervasive in diagnostic laboratories as, despite significant cost of the instrument and its maintenance, consumables and running cost are much lower when compared to conventional methods.

Reviewed Chloe Barnett, BSc



Further Reading

  • All Mass Spectrometry Content
  • What is Mass Spectrometry?
  • Mass Spectrometry Applications?
  • Using Mass Spectrometry for Protein Complex Analysis
  • Coupling and Ionization in Miniature Mass Spectrometry

Last Updated: Nov 26, 2018

Written by

Dr. Tomislav Meštrović

Dr. Tomislav Meštrović is a medical doctor (MD) with a Ph.D. in biomedical and health sciences, specialist in the field of clinical microbiology, and an Assistant Professor at Croatia's youngest university – University North. In addition to his interest in clinical, research and lecturing activities, his immense passion for medical writing and scientific communication goes back to his student days. He enjoys contributing back to the community. In his spare time, Tomislav is a movie buff and an avid traveler.

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