MALDI-TOF mass spectrometry (2023)

Mass spectrometry is an analytical technique in which samples are ionized into charged molecules and their mass-to-charge ratio (m/z) can be measured. In MALDI-TOF mass spectrometry, the ion source is a matrix-assisted laser desorption/ionization (MALDI) and the mass analyzer is a time-of-flight (TOF) analyzer.


MALDI is a soft ionization in which a laser strikes a matrix of small molecules to vaporize the analyte molecules without fragmenting or breaking them down. Some biomolecules are too large and can break down when heated, and traditional techniques will fragment or destroy macromolecules. MALDI is suitable for the analysis of biomolecules such as peptides, lipids, saccharides or other organic macromolecules.

MALDI-TOF mass spectrometry (1)

Figure 1. Ionization of analytes by MALDI

  • The MALDI principle

In Figure 1, the analyte is embedded in a very large excess of a matrix compound deposited on a solid surface, called a target, which is typically made of a conductive metal and has points for applying several different samples. After a very short laser pulse, the irradiated spot is quickly heated up and excited to vibrate. The matrix molecules are energetically removed from the sample surface, absorb the laser energy, and transport the analyte molecules with them into the gas phase. During the ablation process, the analyte molecules typically ionize by protonating or deprotonating with neighboring matrix molecules. The most common MALDI ionization format is that the analyte molecules carry a single positive charge.

(Video) MALDI-TOF MS | What are the applications of MALDI-TOF? | Mass spectrometry.

  • Types of laser commonly used in MALDI

Both ultraviolet (UV) and infrared (IR) wavelength lasers are used, but UV lasers are by far the most important light sources in analytical MALDI. Among these, nitrogen lasers and frequency tripled or quadrupled Nd:Yag lasers often serve the majority of applications. IR-MALDI is dominated by Er:Yag lasers, while TEA-CO2 lasers are rarely used.

  • Commonly used MALDI matrix substance

It is believed that the main function of the matrix is ​​essentially to dilute and isolate the analyte molecules from each other. This occurs during the evaporation of the solvent and the simultaneous formation of a solid solution. Then, after laser irradiation, it acts as a mediator for energy absorption. Choosing the correct matrix is ​​the key to success in MALDI. In general, highly polar analytes work best with highly polar matrices, and nonpolar analytes are preferably combined with nonpolar matrices. As shown in Table 1, various matrices have been widely sought and used. Currently, the most widely used matrices are α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid and 2,6-dihydroxyacetophenone.

Tabla 1. Matrices UV-MALDI (Gross J.H., 2006)

nicotinic acidHEPeptides, Proteins
picolinic acidPennsylvaniaOligonucleotides, DNA
3-hydroxypicolinic acidHPA, 3-HPAOligonucleotides, DNA
3-aminopicolinic acid3-ERAOligonucleotides, DNA
6-Aza-2-thiotimineAOligonucleotides, DNA
2,5-dihydroxybenzoic acidDHBProtein, Oligosaccharide
DHB-based mixesDHB/XY y Super-DHBProtein, Oligosaccharide
α-cyano-4-hydroxycinnamic acidα-CHC, α-CHCA, 4-HCCA, CHCAPeptides, smaller proteins, triacylglycerols, many other compounds
4-chloro-α-cyanocinnamic acidCLICKpeptide
2-(4-hydroxyphenylazo)benzoesauriumLANGUAGEPeptides, proteins, glycoproteins, polystyrene
2-mercaptobenzotiazolMBTPeptides, proteins, synthetic polymers
5-cloro-2-mercaptobenzotiazolCMBTglycopeptides, phosphopeptides and proteins
of 2,6-dihydroxyacetophenoneDHAPGlycopeptide, Phosphopeptide, Protein
2,4,6-trihydroxyacetophenoneGRACIASSolid State Supported Oligonucleotides
Ditranol (1,8,9-antracentriol)nonesynthetic polymers
9-nitroantraceno9-NAfullerenes and derivatives
Benzo[a]pirenononefullerenes and derivatives
2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malonitrileDCTBOligomers, polymers, dendrimers, small molecules

Time of Flight (TOF) Analyzer

MALDI-TOF mass spectrometry (2)

Figure 2. General scheme of the TOF analyzer. (A) TOF coating analyzer; (B) reflector TOF analyzer; (C) The process of deriving the time it takes for ions to pass through the field-free region in the coating TOF analyzer.

(Video) Theory of MALDI-TOF Mass Spectrometry

  • The TOF principle

As shown in Figure 2, the basic principle of TOF is that ions with different m/z are temporarily scattered during their flight along a fieldless drift path of known length. As long as all the ions start their journey at the same time, or at least with a short enough time interval, the lighter ones will reach the detector before the heavier ones.

  • Liner TOF Analyzer and Reflectron TOF Analyzer

In theory, all ions get the same initial kinetic energy, so after traveling through the field-free region, the ions arrive at the detector with the same m/z at that time. In practice, however, the impulse is not felt by all ions to the same intensity, and therefore not all ions with the same values ​​of m/z reach their ideal velocity. To correct this problem, a reflection is often applied to the end of the drift zone. The reflectron consists of an array of high-voltage ring electrodes that can normally repel ions at a slightly offset angle along the length of the flight tube.

Ions of different kinetic energies penetrate the reflectron at different depths before being ejected from the reflectron in the opposite direction. Faster ions with more kinetic energy travel a longer distance than slower ones and therefore spend more time in the reflectron than slower ions with less energy. In this way, the detector receives ions of the same mass (approximately) at the same time. As a result, this design for TOF mass analyzers has significantly increased their resolution. However, the Reflectron TOF analyzer is not suitable for analytes that are not stable enough to survive the electric field.

The MALDI-TOF Mass Spectrometry Process

MALDI-TOF mass spectrometry (3)

Figure 3. The MALDI-TOF mass spectrometry process (Clark A.E.,et al.; 2013)

(Video) MALDI TOF Process

The analyte should be soluble in any solvent to at least about 0.1 mg/mL. And the matrix dissolves to give a saturated solution or a concentration of about 10 mg/ml. The analyte solution is then mixed with the matrix. To obtain optimized MALDI spectra, the matrix to analyte molar ratio is typically adjusted to be within the range of 1,000:1 to 100,000:1. The mixture is then placed on a metal target plate for analysis. After drying, the sample-matrix mixture crystallizes to form a solid sample deposit embedded in the matrix. The plate is then loaded into the MALDI-TOF instrument and analyzed using dedicated software for each system. MALDI leads to sublimation and ionization of both the sample and the matrix. These generated ions are separated based on m/z using a TOF analyzer and a spectral representation of these ions is generated and analyzed by the MS software, producing an MS profile.

Application of MALDI-TOF mass spectrometry

  • Determination of intact mass

Determining the intact mass is fundamental and important for protein characterization, since the correct molecular weight of a protein can indicate the intact structure. MALDI, a mild ionization technique, is suitable for proteins that tend to be fragile and fragment when ionized by other ionization methods. The performance of MALDI-TOF MS is less affected by buffer components, detergents, and contaminants. In addition, it allows determination of intact protein mass with sufficient precision (≤ 500 ppm) for sequence validation. After protein digestion, MALDI-TOF MS can also be used to analyze the obtained peptides for further confirmation of the primary sequence by peptide mass fingerprinting.

  • Peptide-mass fingerprinting (PMF)

MALDI-TOF mass spectrometry has easy operation, good mass accuracy, and high resolution and sensitivity. Therefore, it is widely used in proteomics to identify proteins from simple mixtures through a method called peptide mass fingerprinting, which is often used with two-dimensional (2-DE) gel electrophoresis. In this approach, peptides are generated by digesting proteins of interest with a sequence-specific enzyme such as trypsin. And then the peptides are analyzed by MALDI-TOF mass spectrometry to obtain peptide masses. Experimental masses are compared to a database containing theoretical peptide masses from a given organism with the same sequence-specific protease.

  • Post-Source Decomposition (PSD) MALDI-TOF Analysis

MALDI-TOF mass spectrometers equipped with reflectrons can analyze ion fragments generated from precursor ions that spontaneously decay during flight. Such ions are commonly known as metastable ions, and the decay process in the field-free region between the ion source and the reflectron is commonly known as PSD. PSD fragment ions form within the field-free region before entering the reflectron. PSD fragment ions can be separated, collected, and recorded in the detector by continuously changing the reflector voltage to form a PSD mass spectrum that provides very rich and effective structural information for the primary structure of peptides and proteins. In proteomics study, some protein samples separated by 2DE cannot be identified by PMF or the identification results are not clear. The PSD sequencing function can be applied to the identification of these proteins. Using PSD spectroscopy combined with a database search, proteins can be identified rapidly and with high specificity.

  • Oligonucleotide analysis

With the development of molecular biology techniques and antisense nucleic acid drug technologies, more and more oligonucleotide fragments have been synthesized for use as primers, probes, and antisense drugs. It is absolutely necessary to quickly detect these fragments to determine if the synthesis is complete and if the synthesized sequence is correct. Mass spectrometry, including MALDI-TOF-MS, is by far the best means of doing this. Oligonucleotide analysis using MALDI-TOF-MS was simple, fast, accurate and sensitive, which can be used to determine the complete oligonucleotide sequence.

(Video) MALDI-TOF Mass Spectrometry Explained

  • maldi images

MALDI-TOF can be used to profile and image proteins directly from thin sections of tissue, known as MALDI Imaging Mass Spectrometry (MALDI-IMS). It provides specific information about the local molecular composition, relative abundance, and spatial distribution of peptides and proteins in the analyzed section. MALDI-IMS can analyze multiple unknown compounds in biological tissue sections simultaneously through a single measurement that can obtain a molecular map of the tissue while maintaining the integrity of the cells and molecules in the tissues.

MALDI-TOF mass spectrometry can analyze a variety of biomolecules such as peptides, proteins, carbohydrates, oligonucleotides, etc. Because the ions formed have low internal energy, a major advantage of MALDI-TOF is that the smooth ionization process allows observation of ionized molecules with low fragmentation of analytes, thus allowing even molecular ions of analytes to be identified within the mixes. And it's easy to use and maintain with fast data collection. Selection of the proper matrix substance is important to the success of MALDI-TOF mass spectrometry.

At Creative Proteomics we can offer several services based on our advanced MALDI-TOF mass spectrometry platforms, including:

  • determination of molecular mass
  • Peptide mass fingerprinting (PMF)
  • Imaging Mass Spectrometry (IMS)


1. Gross J H. Mass spectrometry: a textbook. Springer Science and Business Media, 2006.
2. Boesl U. Time-of-Flight Mass Spectrometry: Introduction to Basics. Mass Spectrometry Reviews, 2017, 36(1): 86-109.
3. Guerrera I C, Kleiner O. Application of mass spectrometry in proteomics. Informes de Biociencia, 2005, 25(1-2): 71-93.
4. Fuchs B, Schiller J. Application of MALD-TOF mass spectrometry in lipidomics. European Journal of Lipid Science and Technology, 2009, 111(1): 83-98.
5. Duncan M.W., Roder H., Hunsucker S.W.. Matrix-assisted quantitative laser desorption/ionization mass spectrometry. Reports on Functional Genomics and Proteomics, 2008, 7(5): 355-370.
6. Kenny D. J., Brown J. M., Palmer M. E.,et al. A parallel approach to post-source decomposition MALDI-TOF analysis. Journal of the American Society for Mass Spectrometry, 2006, 17(1): 60-66.

* For research use only. It should not be used in diagnostic procedures.

(Video) Sample Preparation for MALDI-TOF Mass Spectrometry

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What are the limitations of MALDI-TOF? ›

For organisms commonly encountered in the clinical laboratory, MALDI-TOF MS can accurately identify most closely related species. However, there are some exceptions. The inability to discriminate between related species can be due to the inherent similarity of the organisms themselves.

What are the 4 steps in MALDI-TOF? ›

Here are the basics of how this powerful technique works.
  • The Machinery. ...
  • The Matrix. ...
  • Protein Identification. ...
  • SELDI-TOF. ...
  • Increasing the Resolution.

How many organisms can MALDI-TOF identify? ›

For Gram-negative bacteria evaluation, there are 2263 clinical significant species can be analyzed by MALDI-TOF MS identification. Faron et al. showed that MALDI-TOF MS system correctly identified 99.8% (2258/2263) to genus and 98.2% (2222/2263) to species level of all isolates from multiple centers [29].

How do you analyze MALDI-TOF? ›

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. A few TOF analyzers incorporate an ion mirror at the rear end of the flight tube, which serves to reflect back ions through the flight tube to a detector.

What is the accuracy of MALDI-TOF? ›

The MALDI-TOF MS correctly identified 92% of the M. tuberculosis isolates (95% CI of 0.87 to 0.96), and 68% of M. bovisisolates (95% CI of 27% to 100%) to the species level.

Is MALDI-TOF inaccurate? ›

MALDI-TOF MS demonstrated high accuracy for the direct identification of pathogens from urine samples, with a pooled sensitivity of 0.85 and a pooled specificity of 0.93.

What are the principles of MALDI-TOF mass spectrometry? ›

The principle of MALDI

During the ablation process, the analyte molecules are usually ionized by being protonated or deprotonated with the nearby matrix molecules. The most common MALDI ionization format is for analyte molecules to carry a single positive charge.

How does MALDI-TOF identify proteins? ›

One of the main uses of MALDI-TOF-MS is in the identification of proteins, by peptide mass fingerprinting (PMF). Here we describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by 1D or 2D gel electrophoresis can be identified at femtomole levels.

What is MALDI explain how it works? ›

MALDI is the abbreviation for "Matrix Assisted Laser Desorption/Ionization." The sample for MALDI is uniformly mixed in a large quantity of matrix. The matrix absorbs the ultraviolet light (nitrogen laser light, wavelength 337 nm) and converts it to heat energy.

What is the limitation of mass spectrometry? ›

The disadvantages of mass spec are that identifying hydrocarbons that produce similar ions is not very good and it is not able to separate optical and geometric isomers. The disadvantages are offset by combining MS with other methods , for example gas chromatography.

How much sample do you need for MALDI? ›

MALDI Sample Preparation

We will usually need ~5-10 µL. It is best to remove buffer salts and detergents (e.g. by dialysis) prior to analysis and to dissolve the sample in a suitable solvent (e.g. 0.1% TFA/water) which will not degrade the spectrum.

What molecule does MALDI-TOF identify? ›

MALDI-TOF MS detects many different biomolecules, such as nucleic acids, peptides, proteins, sugars and small molecules. This technology identifies microorganisms via the generation of fingerprints of highly abundant proteins followed by correlation to reference spectra in a database.

What makes a good matrix for MALDI? ›

Your matrix should have a strong optical absorption in either the ultraviolet or infrared range, in order to rapidly and efficiently absorb the laser irradiation. The matrix needs to contain polar groups, which allow them to be used in aqueous solutions.

What is a MALDI score? ›

The MALDI Biotyper system classifies species identification according to log scores: mass spectra yielding a log score above 2.0 are assigned the label 'highly confidence identification', whereas spectra with a log score between 1.7 and 2.0 are assigned 'low confidence identification'.

What would be the advantage of using MALDI-TOF in a clinical setting? ›

The use of MALDI-TOF MS in the medical lab reduces the time between specimen collection and diagnosis. Also, preexamination processing of organisms for analysis by MALDI-TOF MS is technically simple and reproducible. Another benefit of this technology is accuracy.

Does MALDI-TOF require culture? ›

MALDI-TOF is routinely used in microbiology laboratories to identify microorganisms suspected of causing infections in patients. Patient-derived biosamples are cultured, and a colony is spotted onto the MALDI target plate, treated with formic acid and matrix, and analyzed by the MALDI-TOF instrument.

Why MALDI is suitable for biological samples? ›

In the last two decades, MALDI TOF MS — matrix-assisted laser desorption mass spectrometry, proved to be an especially convenient tool for these analyses. The main advantages of this method are its rapidity and high sensitivity which is particularly appreciated in the case of studies of complex biological specimen.

Is MALDI-TOF high throughput? ›

MALDI-TOF MS is a powerful analytical technique that provides a fast and label-free readout for in vitro assays in the high-throughput screening (HTS) environment.

What is the sensitivity and specificity of MALDI-TOF? ›

The species identification by MALDI-TOF MS yielded sensitivity at 97.9% [96.2%–99.6%] and specificity at 69.5% [61.9%–77.1%] for A. baumannii differentiation (Table 1).

How accurate is mass spectrometry? ›

Modern mass spectrometers generally report accurate mass measurements to four decimal places (seven significant figures for masses between 100 and 999 Da) and sometimes more.

Does MALDI cause fragmentation? ›

It is well known experimentally that MALDI matrices can cause more or less analyte fragmentation, and matrices are therefore characterized as “hard” or “soft”, respectively.

What are the applications of MALDI-TOF? ›

The MALDI-TOF MS system is increasingly used in clinical diagnostic laboratory for viral identification, mutation analysis, genotyping, and antiviral resistance studies.

Why is MALDI-TOF MS particularly important in clinical microbiology? ›

Using standardized procedures, the resolution of MALDI-TOF MS allows accurate identification at the species level of most Gram-positive and Gram-negative bacterial strains with the exception of a few difficult strains that require more attention and further development of the method.

Why is MALDI used for imaging? ›

One main advantage of MALDI-MSI over other imaging modalities is its ability to determine the spatial distribution of hundreds of analytes within a single imaging run, without the need for a label or any prior knowledge (Schnackenberg et al., 2021).

What are the advantages of MALDI-TOF TOF? ›

Benefits and limitations

MALDI-TOF MS is robust, cost-effective and able to provide rapid and accurate identification of micro-organisms from culture despite the potential spectral variation introduced by different culture conditions, protein extraction methods and different laboratory processes.

Why is MALDI useful in analysis of protein and peptides? ›

Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) is a valuable tool for the analysis of peptides and proteins. Particularly useful features include high sensitivity, fast data acquisition, ease of use, and robust instrumentation.

What is MALDI technique in mass spectrometry? ›

In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation.

What are the strengths and weaknesses of mass spectrometry? ›

It is an excellent tool for identifying unknown components in a sample or confirming their presence. Disadvantages of mass spec are that it isn't very good at identifying hydrocarbons that produce similar ions and it's unable to tell optical and geometrical isomers apart.

What affects sensitivity of mass spectrometry? ›

Studies have shown that the gas expansion in high vacuum has a great influence on ion transfer and mass analysis, thereby affecting the sensitivity.

How can I improve my mass spectrometry? ›

In summary, there are four basic strategies used on mass analyzers for the improvement of mass spectrometers' sensitivity, including the improvement of ion transmission efficiency, the selective enrichment of targeted ions, the improvement of the ion utilization rate and the improvement of the signal-to-noise ratio (S/ ...

How much sample do you need for mass spectrometry? ›

The amount of sample required depends on the mass spectrometry service to be performed. In general, 1 mL of a 10 ppm sample or 1 mg in solid form is enough for all applications.

How to prepare samples for MALDI-TOF? ›

►► Sample preparation

Mix 1 part saturated HCCA solution with 1 part sample solution. 2. Deposit 0.5 ตL of the matrix/analyte mixture onto the MALDI target and allow to dry. The concentration of the peptide/protein solution should be between 10 fmol – 1 pmol/µL.

What is the minimum bacterial CFU count required for Maldi-TOF MS? ›

MALDI-TOF MS requires high bacterial counts to provide reliable scores. Inoculation with at least 1.0 × 105 CFU/ml was required to obtain reliable scores for E.

Can MALDI be used for small molecules? ›

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) does not work efficiently on small molecules (usually with molecular weight below 500 Da) because of the interference of matrix-related peaks in low m/z region.

How to prepare matrix for MALDI? ›

1. Dissolve one tube of MALDI matrix with 100 μl of 70/30 acetonitrile/water with 0.1% TFA, which results in 10 mg/ml of CHCA or SA or 40 mg/ml of DHB. 2. Apply 0.5 μl of a protein digest (0.1-1 pmol/μl) onto a MALDI target plate and immediately add 0.5 μl of the MALDI matrix solution.

What is the first matrix utilized for MALDI? ›

A matrix material composed of alpha-cyano-4-hydroxycinnamic acid or CHCA is typically used for peptide analysis using MALDI. MALDI is also suitable for direct analysis of peptides spotted on a MALDI target and, in general, will provide higher quality data than that generated from an in-gel digestion.

Why is it important to matrix match? ›

Matrix matching is used in analysis to compensate for matrix effects that influence analytical response. It has been a widely discussed topic in electro-spray mass spectrometry where the ionization suppression is a major problem in accurate quantitative analysis.

What are the limitations of spectrometry? ›

The main disadvantage of mass spectrometry is that it is costly, need a skilled technician, and it is not a portable system. We will unable to differentiate among isomers of the molecule with the same charge-to-mass ratio. Chiral columns may be required to separate enantiomers.

Is MALDI destructive? ›

Therefore, MALDI mass spectrometry can also be used for the analysis of biological macromolecules. Compared with the hard ionization methods of early mass spectrometry, soft ionization techniques including ESI and MALDI are less destructive to sample molecules and can retain the integrity of the entire molecule.

Which of the following Cannot be used as a maldi matrix compound? ›

10. Which of the following cannot be used as a MALDI matrix compound? Explanation: 2,3-Dihydroxybenzoic acid cannot be used as a MALDI matrix compound. α-Cyano-4-hydroxycinnamic acid, sinapinic acid, and 3-Hydroxypicolinic acid can be used as a MALDI matrix compound.

What are two drawbacks of mass spectrometry? ›

1. The main disadvantage of mass spectroscopy is that those hydrocarbons which produce similar ions are not identified. 2. It is also not able to separate optical and geometrical isomers.

What are the sources of error in using spectrometer? ›

In practice there are other sources of error, such as environmental effects on photometer and sample, temperature, line voltage fluctuations, vibrations, contamination, or heating of the sample by the photometer. All these factors may impair the measured result, and ways and means are known to test and eliminate them.

Why is MALDI-TOF good? ›

Due to the fact that formed ions have low internal energy, a great advantage of MALDI-TOF is that the process of soft-ionization enables observation of ionized molecules with little to fragmentation of analytes, allowing the molecular ions of analytes to be identified, even within mixtures.

Is MALDI a soft technique? ›

MALDI is a soft ionization technique in which a laser energy-absorbing matrix is used to create ions.

What type of laser is used in MALDI-TOF? ›

Traditionally Nitrogen lasers (337 nm) have been used as a source for MALDI-TOF but more recently frequency tripled solid state lasers (355 nm) have become more common due to their reliability and lifetime.

How long does it take to run MALDI? ›

In our experience, teaching the use of MALDI-TOF MS to laboratory technician personnel, including the protein extraction procedure, requires only about 1 h.


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