MALDI-TOF Mass Spectrometry in the Biosciences

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) spectrometry is a powerful analytical technique that is widely used in both clinical and laboratory settings.1

Scientist putting a sample in a test tube in a chemical laboratory

Image Credit: Minerva Studio/

The scientific significance and wider impact of the development of laser desorption techniques in combination with mass spectrometry was recognized with the 2002 Nobel Prize in Chemistry.2 Since then, there have been many advances in both the hardware for performing these experiments and associated data analysis methods that have led to a much more widespread adoption of MALDI-TOF-based methods.

Principles of MALDI-TOF Mass Spectrometry

MALDI refers specifically to the ionization or desorption method that is used to introduce the analyte into the spectrometer. One of the most common mass spectrometry ionization techniques is electron impact, where the sample is bombarded with electrons to induce ionization.3 Most mass spectrometer designs rely on the analytes being charged species so their flight paths can be deflected and controlled using magnetic fields.

The problem for very high molecular weight species and more ‘fragile’ molecules like biomolecules is that electron impact ionization can also lead to a large amount of fragmentation. While this can be useful as fragmentation patterns can also be very informative for chemical identification, excess fragmentation can make it hard to identify the total molecular weight of a species or lead to too much spectral congestion.

MALDI is a type of ‘soft’ ionization technique where the analyte is prepared in a matrix that absorbs a particular wavelength of light. A laser is then used to irradiate the matrix with a wavelength it absorbs, causing rapid heating and vaporization of the matrix. As the matrix is vaporized, the analyte is released and becomes charged via different mechanisms and, as a result, can be detected.4

From there, the charged analytes are extracted into the mass analyzer and the length of time it takes for each analyte to fly through the time-of-flight region to the detector is measured. As the flight time for the analytes is dependent on the mass to charge (m/z) ratio, the different arrival times can be used to discriminate between different species. 

Instrumentation and Workflow

There are now a number of commercial solutions available for MALDI-TOF instruments. A general instrument will involve the ionization region and laser source, then a series of lenses to extract the charged analytes and inject them into the field-free TOF region, with more lenses to accelerate the charged particles into the detector.

MALDI-TOF is a relatively mature technology and most of the recent focus has been on the development of improved data analysis routines, but there are still some optimization of acquisition settings that can be performed to help apply MALDI TOF to the analysis of a larger number of organisms.5

There is still some work to be done in standardizing MALDI TOF workflows in the analysis of species such as fungi, but a typical routine will involve preparation of the analytes and optimization of the matrix, measurement of references, identification of analytes in a sample of interest and then spectral and statistical analysis.6


Key application areas of MALDI-TOF include biomedical sciences1, forensics7 and any application where species identification is necessary, particularly if this is part of more complex mixtures. More recently, MALDI-TOF has become a popular method for environmental studies looking to isolate and identify environmental bacteria owing to the high accuracy and precision of identification.8

What makes MALDI-TOF mass spectrometry so useful in biosciences is that many of the species being detected have very high molecular weights and are prone to extensive fragmentation. For viral studies, MALDI-TOF mass spectrometry is compatible with the detection of a wide number of specimens and has a much faster turnaround time than processes like PCR for the identification of species.9 MALDI-TOF mass spectrometry also has the advantage of detecting multiple viral species in co-infected samples.

Advantages and Disadvantages

The key appeal for many analytical applications of MALDI-TOF is the relatively rapid analysis times. Particularly with more extensive spectral databases making spectral matching and identification more straightforward, a MALDI-TOF experiment can sometimes be run in as little as a few minutes.

There can be some challenges in the sample preparation, in particular, finding compatible matrices that do not interfere with the spectral features of interest, but as large numbers of clinical microbiology studies are performed, there is greater availability of operating procedures for the measurement of different pathogen types.10

Future Developments

For clinical applications and rapid microbiological species identification, rigorous validation of the MALDI-TOF measurement techniques on well-characterized clinical species still needs to be done.10 However, it is clear that MALDI-TOF mass spectrometry has been used with great success and cross-technique validation on a number of different sample types already.

One of the biggest areas of development for rapid diagnostics with MALDI-TOF mass spectrometry is the use of algorithms to accelerate the data analysis of the resulting spectra. Screening for biomarkers and performing measurements for antimicrobial susceptibility testing all require understanding what species are related to the pathogen or disease of interest, which can be very complex in the types of samples analyzed with MALDI-TOF but automation can help identify multiple fingerprints in spectra and also perform correlation analysis for such testing.11 ​​​​​​​


  1. Li, D., Yi, J., Han, G., & Qiao, L. (2022). MALDI-TOF Mass Spectrometry in Clinical Analysis and Research. ACS Measurement Science Au, 2, 385–404.
  2., Nobel Prize Outreach, 2024, (accessed March 2024)
  3. El-aneed, A., Cohen, A., Banoub, J., El-aneed, A., Cohen, A., & Banoub, J. (2009). Mass Spectrometry , Review of the Basics : Electrospray , MALDI , and Commonly Used Mass Analyzers Mass Spectrometry , Review of the Basics : Applied Spectroscopy Reviews, 44, 210–230.
  4. Zenobi, R., & Knochenmuss, R. (1999). Ion Formation in Maldi Mass Spectrometry. Mass Spectrometry Reviews, 17, 337–366. tps://<337::AID-MAS2>3.0.CO;2-S
  5. Nellessen, C. M., & Nehl, D. B. (2023). An easy adjustment of instrument settings (‘ Peak MALDI ’) improves identification of organisms by MALDI ‑ ToF mass spectrometry. Scientific Reports, 1–8.
  6. Barker, K. R., Kus, J. V, Normand, A., Gharabaghi, F., Mctaggart, L., Rotstein, C., Richardson, S. E., & Campigotto, A. (2022). A Practical Work fl ow for the Identi fi cation of Aspergillus , Fusarium , Mucorales by MALDI-TOF MS : Database , Medium , and Incubation Optimization. Journal of Clinical Microbiology, 60(12), 1–14.
  7. Groeneveld, G., Puit, M. De, Bleay, S., Bradshaw, R., & Francese, S. (2015). Detection and mapping of illicit drugs and their metabolites in fingermarks by MALDI MS and compatibility with forensic techniques. Scientific Reports, 5, 11716.
  8. Ashfaq, M. Y., Da, D. A., & Al-ghouti, M. A. (2022). Application of MALDI-TOF MS for identification of environmental bacteria : A review. Journal of Environmental Management, 305, 114359.
  9. Camarasa, C. G., & Cobo, F. (2018). Application of MALDI-TOF Mass Spectrometry in Clinical Virology. In The Use of Mass Spectrometry Techonology (MALDI-TOF) in Clinical Microbiology (pp. 167–180).
  10. Feucherolles, M., Poppert, S., Utzinger, J., & Becker, S. L. (2019). MALDI ‑ TOF mass spectrometry as a diagnostic tool in human and veterinary helminthology : a systematic review. Parasites & Vectors, 1–13.
  11. Zhu, Y., & Girault, H. H. (2023). Algorithms push forward the application of MALDI – TOF mass fingerprinting in rapid precise diagnosis. VIEW, 4, 20220042.

Last Updated: Mar 19, 2024

Rebecca Ingle, Ph.D

Written by

Rebecca Ingle, Ph.D

Dr. Rebecca Ingle is a researcher in the field of ultrafast spectroscopy, where she specializes in using X-ray and optical spectroscopies to track precisely what happens during light-triggered chemical reactions.


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