MALDI-TOF Mass Spectrometry: Principle, Applications in Microbiology

MALDI-TOF mass spectrometry is a versatile analytical technique to detect and characterize mixtures of organic molecules. In microbiology, it is used as a rapid, accurate, and cost-effective method for identifying microorganisms (bacteria, fungi, and viruses). Identification of the organisms by MALDI-TOF (MS) is based on assessing protein profiles and database comparison. A typical experiment consists of the growth of the organism (e.g., bacteria), colony selection and placement on a target, addition of matrix, and analysis with MALDI-TOF MS.

Matrix is a small organic molecule used to facilitate ionization process by absorption of UV light.

MALDI-TOF Mass Spectrometer

MALDI stands for Matrix-Assisted Laser Desorption Ionization. In this ionization method samples are fixed in a crystalline matrix and are bombarded by a laser. The sample molecules vaporize into the vacuum while being ionized at the same time without fragmenting or decomposing.

TOF stands for Time oFlight, a mass spectrometry method that separates ions by their mass to charge ratio and determines that mass to charge ratio by the time it takes for the ions to reach a detector.

This technology generates characteristic mass spectral fingerprints which are compared with large library of mass spectra. As the spectral fingerprints are unique signatures for each microorganism accurate microbial identification at the genus and species levels is done using bioinformatics pattern profiling. 

Working Principle of MALDI-TOF Mass Spectrometry 

The MALDI TOF process is a two-phase procedure;

  1. Ionization Phase
  2. Time of Flight Phase

Ionization Phase:
Initially, the samples are fixed in a crystalline matrix on a target plate and are bombarded by a laser. The sample molecules vaporize into the vacuum while being ionized at the same time. High voltage is then applied to accelerate the charged particles.

The second step is the time-of-flight mass spectrometry phase.

  1. In the linear mode, particles will impinge upon the linear detector within a few nanoseconds after ionization. Higher mass molecules will arrive later than lighter ones. Flight time measurement makes it possible to determine molecule masses directly. Each peak in the spectrum corresponds to the specific mass of the particle along the time axis, starting with the ionization moment.
  2. In the reflector mode, the particles are diverted so that they fly towards a second detector. In addition to extending the flight distance, the reflector also focuses on the masses. The combination of these two effects makes for higher resolution than in the linear mode.

The net result is a generation of a mass spectrum that is compared with those of well-characterized organisms available in the reference library database to identify the isolate.

Proteomic Fingerprints of Microorganisms (

Multiple commercial  microbial identification platforms are available such as VITEK® MS system of BioMérieux, BD Bruker MALDI Biotyper System, Andromas etc. 

MALDI-TOF Operating Principle (Image source: Cheikh Ibrahima Lo)


  1. Pick a bacterial colony and smear it onto a target plate.
  2. Add 1-2 µl of a matrix consisting α-Cyano-4-hydroxycinnamic acid (CHCA) dissolved in acetonitrile (50%) and 2.5% trifluoroacetic acid onto it and dry it on the target plate (at room air)
  3. Place the target plate into the plating chamber of the mass spectrometer, close it and perform the analysis.

Target plate is made of polished or ground stainless steel and has spots for several different samples to be applied. Both ready-to-use disposable and reusable MALDI target plates are available.
Typical workflow

Applications in Microbiology

Microbial identification by MALDI-TOF MS has skyrocketed over the last couple of years because it offers species-level identifications in minutes at low costs with accuracy that matches and often exceeds that of conventional identification systems.

MALDI-TOF MS is being used for routine diagnostic or diagnostic-like purposes in a clinic, veterinary, pharma, and food microbiology (food quality control) laboratories as well as for environmental monitoring, biodefense, and various biological research. 

The two major platforms for MALDI-TOF (MS) organism identification are Vitek MS (bioMérieux) and the Biotyper (Bruker Daltonic).

Nisha Rijal Operating VITEK MS
Operating Vitek-MS for rapid identification of microorganisms

Advantages of MALDI-TOF Mass Spectrometry

  • Significantly decreases the turnaround time. Processing time is similar to rapid biochemicals. 
  • The sample preparation is simple, and the sample requirement is minimal.  A single colony is sufficient to generate spectra of sufficient quality.
  • Cost effective-low consumable costs
  • Automated, robust, interlaboratory reproducibility
  • Broad applicability (all types of bacteria, including anaerobes and fungi)
  • Adaptable-open system, expandable by user


  • Identifying new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains.
  • No susceptibility information is provided 
  • Not useful for direct testing of clinical specimens (except urine)
  • Some organisms require repeat analysis and additional processing (extraction)
  • The acceptable score cutoffs vary between studies and some closely related organisms are not differentiated
  • Some organisms currently cannot be reliably identified by this method, such as Shigella spp and Streptococcus pneumoniae

References and Further Reading

  1. Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology
  2. MALDI Biotyper system of Bruker.
  3. The role of MALDI-TOF in Clinical Microbiology
  4. VITEK-MS system of BioMérieux

Nisha Rijal

I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.

5 thoughts on “MALDI-TOF Mass Spectrometry: Principle, Applications in Microbiology

  1. Good write up you have here.
    Please i will appreciate it if you make part of your write up to contain bioinformatic, molecular genetics.

    Good work prof.

    1. Thank you so much for pointing out the typo and helping us to improve the content quality in microbeonline.

  2. MALDI-TOF is a technological marvel to me. I worked in medical micro labs in the 70’s and 80’s. This is when the Vitek was new. This is fascinating stuff.
    Thanks so much for your blog. I love it.

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