Microbial Identification by MALDI-TOF-MS: An advanced, Rapid and High Throughput Identification system

Microbial Identification by  MALDI-TOF-MS: An advanced, Rapid and High Throughput Identification system


  • Post By :

  • Source: Microbioz India

  • Date: 09 May,2023

Most of the MALDI-TOF-MS Microbial ID providers have a facility to update the database periodically and create their own database of their own isolates. Unlike other phenotypic identification methods, the database is continuously updated and meticulously enhanced. The exposure risk to microbiologists is low. This is because samples are inactivated by extraction before use.


Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a powerful technology used for the identification of microorganisms. Though the system is “phenotypic‟, it, in some senses, connects the gap between the reliability of the results based on a biochemical based phenotypic system and a genotypic based identification. The system is also very fast, making it a good example of a ‘rapid microbiological method’.

In 1985, Franz Hillen Kamp and Michael Karas, both from the University of Frankfurt, published a paper in which they described the use of MALDI-TOF-MS to analyze peptides. This technique uses a laser to ionize molecules embedded in a matrix, creating charged particles that can be detected according to their mass-to-charge ratios. The data is recorded as mass spectrum. This approach allows for the accurate determination of molecular weight.

In 1996, researchers at the University of Zurich were the first to demonstrate the use of MALDI-TOF-MS for the identification of microorganisms based on their unique protein profiles. This breakthrough sparked interest in using it for the rapid identification of microorganisms.

Over the next decade, numerous studies were conducted to evaluate its performance for microbial identification. These studies showed that MALDI-TOF-MS could accurately identify a wide range of bacteria, fungi, & viruses, and was faster as well as more accurate than traditional methods. In 2009, Bruker Daltonics introduced the commercial MALDI Biotyper for microbial identification.

It quickly became widely adopted by clinical microbiology laboratories. MALDI-TOF-MS has continued to evolve and improve. Advances in sample preparation, data analysis, and reference databases have led to increased accuracy and expanded capabilities. Today, MALDI-TOF-MS is widely recognized as a reliable, rapid, and cost-effective method for microbial identification, with applications in clinical microbiology, food safety, environmental monitoring, and more.

Principle of working of MALDI-TOF-MS:

How does MALDI TOF mass spectrometry work for bacterial identification and fungal characterization? The sample for analysis by MALDI TOF MS is prepared by mixing with a solution of an energy-absorbent, organic compound called a matrix (for example benzoic acid or cinnamic acid derivatives, liquid crystalline matrices).

When the matrix crystallizes on drying, the sample (microbial proteins in the samples) entrapped within the matrix also co-crystallizes. The sample within the matrix is ionized in an automated mode with a “soft ionization technique‟ by deployment of a short nitrogen laser pulse to ionize the molecule.

Figure 1: MALDI-TOF-MS process

This “mild” ionization means that the formed ions have low internal energy. This allows for the observation of ionized molecules with little or no fragmentation. Desorption and ionization with the laser beam generate singly protonated ions from analytes in the sample. The protonated ions are then accelerated at a fixed potential, where they separate from each other based on their mass-to-charge ratio (m/z). The charged analytes are then detected and measured using a time of flight (TOF) analyzer. For microbiological applications, TOF mass analyzers are mainly used. During MALDI-TOF analysis, the m/z ratio of an ion is measured by determining the time required for it to travel the length of the flight tube towards a mass detector at the top. Based on the TOF information, a characteristic spectrum called peptide mass fingerprint (PMF) is generated for analytes in the sample. This PMF is compared with the PMF in the database and a report is generated.  Microbial ribosomal and some housekeeping proteins are key signature proteins, especially in the region of 2,000 to 20,000 dalton. Based on these principles, the system is a rapid and highly reliable analytical tool for the characterization of diverse microorganisms found in pharmaceutical, bioscience research and healthcare facilities.

The Role of the Matrix:

The matrix acts like a shield between our sample and the laser. Most commonly, matrix is applied to the cells or samples using suitable solvents. This is often done with a mixed solution of highly purified water and an organic solvent, such as acetonitrile/trifluoroacetic acid.

An ideal matrix must show some desirable characteristics.  Firstly, it is expected that the matrix has a low molecular weight, which ensures easy evaporation, but one that is still large enough that it won’t evaporate during sample preparation and will be stable under vacuum. Secondly, the matrix should have a strong optical absorption, to absorb the laser irradiation efficiently. Next, the matrix needs to contain polar groups, which allow them to be used in aqueous solutions. Finally, the matrix typically contains a chromophore, which is a handy visual aid. α-cyano-4-hydroxycinnamic acid (CHCA) and 3,5-dimethoxy-4-hydroxycinnamic acid (DHCA) are a few of the most popular matrix for detection of middle-weight molecules such as peptides/proteins and mostly used in positive ion mode. CHCA is the most preferred for microbial identification.


There are two basic approaches. Typically, pure colonies of log phase growing microorganisms are prepared on an appropriate agar plate. It is recommended that plates are not held at 2-8°C prior to testing. Cold storage can affect the quality of the spectrum. As an alternative, in the clinical setting, clinical specimens, such as blood culture material, can be used with a special extraction kit.

For bacterial samples, a direct coating or extension method is used. For fungal & mycobacterial samples, a protein extraction method is recommended. The target slide is a metal reusable slide with ninety-six test spots (Single-use plates are also available).

During testing of samples, a calibrator is also spotted for calibration of the system. The calibration is done using purified proteins from reference microbes, such as ATCC type strains.

Briefly, a calibrator extracted proteins   or tiny portion of a single bacterial colony was spotted onto a single spot on the target slide to form a homogeneous smear.

Figure 2: Parts of MALDI-TOF & required Lab Accessories

After drying, it is overlayed by 1 µL of the ready-to-use matrix (CHCA). After drying at ambient temperature, the target plate is placed into the ionization chamber of the system. Spots to be analyzed are shot by an ultraviolet nitrogen laser desorbing microbial and matrix molecules from the target plate. The laser, operating at 337 nm, is generated by nitrogen oxide.

Most of the energy is absorbed by the matrix, converting it to an ionized state. Through random collision in the gas phase, charge is transferred from matrix to microbial protein molecules. The cloud of ionized molecules is funneled through a positively charged electrostatic field (20 kV) into the time off-light mass analyzer, a tube under vacuum. The ions travel toward an ion detector with small sized analytes traveling fastest, followed by progressively larger analytes. The pulsed laser takes individual ‘shots’ rather than working in continuous operation.

As ions emerge from the mass analyzer, they collide with an ion detector, generating a mass spectrum representing the number of ions hitting the detector over time. Although separation is by mass-to-charge ratio because the charge is typically single for the described application, separation is effectively by molecular weight. This means that minor ions reach a TOF detector before larger ions. Microbial identification was performed by comparing the generated spectrum from the samples with the reference spectra in the database.

Spectra analysis was collected by Acquirer and analyzed by respective Analyzer software. The final report is generated showing the closest match from the database and interpretation is done based on the score generated by the system. The manufacturer’s interpretation criteria as mentioned in the report was used for ID interpretation.

Key advantages of MALDI-TOF-MS identification:

One important advantage of MALDI-TOF-MS is the rapid time-to-result. With MALDI-TOF, results are generally available within minutes. In keeping with the relatively rapid testing, sample preparation is also quick and straightforward. Compared to classical and genomic methods, minimal skills are required to perform and to have reproducible results. At a given time, 96 microbes can be scanned within 20 min or less minutes and, in continuous mode, 960 microbes can be identified within an hour. Most of the MALDI-TOF-MS Microbial ID providers have a facility to update the database periodically and create their own database of their own isolates. Unlike other phenotypic identification methods, the database is continuously updated and meticulously enhanced. The exposure risk to microbiologists is low. This is because samples are inactivated by extraction before use. The results obtained are robust, accurate, and highly reproducible. The cost of the test per microbe by this method is much lesser as compared to genomic and biochemical methods. It is a unique kind of high through put identification method available today. Finally, its workstation can be connected to other automation systems in microbiology.


  1. Bacterial endospores, may cause spectral interference leading to poor to no identification. This can be overcome by using its younger log phase cultures.
  2. Sometimes, MS spectra cannot readily differentiate closely related organisms (such as Escherichia coli & Shigella species, and different yeasts). In such cases, confirmation is done using selective or differential media.
  3. For any identification system, the reliability in identifying is based on how comprehensive the database is.
  4. MALDI-TOF databases have a clinical bias and need to be strengthened for use with industrial and pharmaceutical microbiology. This can be overcome by selecting a system with a bigger and wider database of microbial species. Also, maintaining our own database may help to reduce this challenge.
  5. Microorganisms producing capsules/slime are more resistant to cell lysis. This can lead to a low extraction yield, lower quality spectra and thus mis- or null-identification. This can be overcome to a large extent by following protein extraction protocols.
  6. The type of media used can lead to interference. The media selected during method verification should be used as standard throughout all subsequent identifications. Any system provider highlights such limitations specifically mentioning such cultures.

Spectrum of the microbial database:

All MALDI-TOF-MS systems can identify a broad spectrum of bacteria, including aerobic & anaerobic bacteria, Gram-positive & negative bacteria, and filamentous fungi & yeasts up to species level. These microbial strains are isolated from different sources such as humans, animals, and the environment. Bacteria regarded as difficult to culture have a high success in being identified using the system. An example is Mycobacteria species. The outcomes are generally reproducible. Recent advanced versions also have validated Software and Kits for Anti-Microbial Resistance (AMR) patterning.

How to select an ideal MALDI-TOF-MS microbial ID system:

First, the daily load of samples is to be identified (1-5 per day, 5-20 per day, 20-50 per day or around 100 per day). Moreover, one must decide if one needs an AMR system as well as identification or only microbial identification.  Having decided that part and now when we want to decide which models to be purchased, there are certain criteria we suggest.  First check how big the database is available in the system. Within the database, the important is how many species there are in the database rather than how many strains.

From a performance consistency point of view, it is appropriate to understand how many million or billion shots of the laser system generated in that MALDI-TOF-MS. The bigger this number, the more is the life of the laser system and very consistent results will be generated. The higher the vacuum pump capacity, the lower is the time to result.

Future scope:

New matrices are constantly being added to the field. It is worth researching what matrix might work for your experiment and talking to experts in the field who have previously used it! Currently, the majority MALDI-TOF and TOF/TOF systems providers offer maximum utility, and their improved dynamic range enables deeper analyses of diverse sample types across a broad mass range.

The ability to quickly analyze samples in both positive and negative ionization modes support application flexibility for the assessment of peptides, proteins, or polymer integrity in samples.

With a laboratory-friendly footprint and the option to upgrade from TOF to TOF/TOF capabilities in the field, many offer true value and analytical versatility, and continue providing innovative solutions for better and faster use of microbial IDs.

About Author