MALDI-TOF Mass Spectrometry: Principle, Applications in Microbiology

Last updated on June 6th, 2021

MALDI-TOF mass spectrometry is a versatile analytical technique to detect and characterize mixtures of organic molecules. In Microbiology, it is being used as a rapid, accurate, and cost-effective method for the identification of microorganisms (bacteria, fungi, and viruses). A typical experiment consists of 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 is 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 in 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 make 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 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 which 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 soure: 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 on to 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 are 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 clinic, veterinary, pharma and food microbiology (food quality control) laboratories as well as for environmental monitoring, biodefense and various biological research. 

Advantages of MALDI-TOF Mass Spectrometry over Conventional Technology

  • 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 in order to generate spectra of sufficient quality
  • Cost effective-low consumable costs
  • Automated, robust, interlaboratory reproducibility
  • Broad applicability (all types of bacteria including anaerobes, fungi)
  • Adaptable-open system, expandable by user


  • Identification of 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
About Acharya Tankeshwar 474 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.