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Posted on September 29, 2022 by Zeng YingLab Equipment

What is MALDI-TOF?

Since 1886, when Goldstein invented the ion source commonly used in early mass spectrometers, to 1942, when the first single-focus mass spectrometer was commercialized MALDI-TOF mass spectrometry was basically in the theoretical development stage. The subsequent development and refinement of MALDI-TOF mass spectrometry in ionization techniques and analytical techniques have led to its rapid application in many fields such as geology, space research, environmental chemistry, organic chemistry, and pharmaceuticals.
MALDI-TOF mass spectrometry is becoming a major supporting technique for protein identification and analysis by determining the mass-to-charge ratio (m/z) of sample ions for compositional and structural analysis. The identification of protein spots separated by two-way gel electrophoresis, which are large in number and small in quantity, is performed by searching the protein database for proteins matching these parameters using various property parameters such as relative molecular mass, isoelectric point, sequence, amino acid composition, peptide mass fingerprinting, etc. If it is not found in the database, it is possible that a new protein has been discovered and sequence analysis is performed to further synthesize DNA probes to express, isolate and identify it.

MALDI-TOF applications


The subsequent development and refinement of MALDI-TOF mass spectrometry in ionization and analytical techniques led to its rapid application in a variety of fields such as geology, space research, environmental chemistry, organic chemistry, and pharmaceuticals. However, even after the emergence of two soft ionization mass spectrometry techniques, plasma desorption (PD) and fast atom bombardment (FAB), the relative molecular masses analyzed by mass spectrometry were only in the range of a few thousand. The real change was represented by the emergence of two new ionization techniques in the mid-1980s: electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), both of which have The high sensitivity and high quality detection range of these two techniques make it possible to detect biomolecules with relative molecular masses up to hundreds of thousands at the fmol (10-15) and even amole (10-18) levels, thus opening up a brand new field of mass spectrometry - biological mass spectrometry, and prompting the widespread application and development of mass spectrometry in the life science field. This has led to the wide application and development of mass spectrometry in the life sciences.

MALDI-TOF for medical applications

MALDI mass spectrometry can provide a rapid and easily solved multi-component analysis method with high sensitivity, selectivity and accuracy, and its scope of application far exceeds that of radioimmunoassay and chemical detection. MALDI-TOF biological mass spectrometry can be mainly used in laboratory medicine for component sequence analysis, structure analysis, molecular weight determination and content determination of each component in living organisms.

Applications of nucleic acid detection

The study of molecular MALDI-TOF of nucleic acids has become one of the most dynamic research directions in the fields of life chemistry, molecular biology and medicine. Through modern biological mass spectrometry, we can not only get the molecular mass of oligonucleotides, but also its sequence information by relevant techniques.

Applications for microbial identification

MALDI-TOF of each bacterial isolate can be used to identify bacteria based on the unique peptide pattern or fingerprint of each bacterium, and Hsu has used tandem mass spectrometry to identify Salmonella J. Because of the high protein content in bacteria, biological mass spectrometry can be commonly used for the identification of bacterial genera, species, and strains; and tandem mass spectrometry can also target the fatty acid composition of sugars or lipids; in addition, the By processing biological samples, tandem mass spectrometry can also detect and identify pathogenic bacteria and spores at the level of single bacteria; analysis of specific lipid components provides insight into the viability and potential infection of pathogenic bacteria in the sample.

Applications of pharmaceutical analysis

The applications of mass spectrometry in pharmaceutical analysis include: synthetic drug component analysis, natural drug composition analysis, amino acid sequence analysis of peptide and protein drugs (including glycoproteins), drug metabolism studies and herbal medicine composition analysis. More applications in laboratory medicine are therapeutic drug monitoring (TDM). Previously, drug detection mainly used immunochemical techniques and high performance liquid chromatography. Although, the immunochemical technique is simple and easy to use, the variety of drugs measured is relatively small. Although the high performance liquid chromatography technique determines more types of drugs, the reliability of characterization is poor. However, liquid chromatography coupled with mass spectrometry (LC.MS) is accurate and rapid for drug detection and can be used for almost all drugs, such as anticancer drugs, immunosuppressants, antibiotics and cardiovascular drugs, and LC.MS technology is expected to be the most powerful tool for drug detection.

What is the principle of MALDI-TOF?

MALDI-TOF mass spectrometry works as follows:
The laser excites the bacteria on the target plate with a substrate that allows the bacterial proteins to fly in a vacuum tube. The detector detects the difference in protein time of flight to create a profile which is then compared to the information in the database to derive the possible species of bacteria.
The MALDI-TOF MS mainframe consists of two parts, the ion source (MALDI) and the time-of-flight (TOF) mass analyzer. The principle of MALDI is to use a laser source to irradiate the sample to be examined with the matrix to form a crystalline film, the matrix absorbs energy from the laser to transfer to the biomolecules, and the ionization process transfers protons to or from the biomolecules to ionize the biomolecules. The principle of TOF is that the ions are accelerated by the electric field to fly through the flight tube, and the m/z (i.e., the m/z of ions is proportional to the flight time of ions) and the signal value are obtained according to the arrival time of the detector and the number of ions, thus forming the corresponding mass spectrometry peak pattern. The spectra obtained on the machine can be compared with the reference common in the database to obtain the closest strains, and the corresponding identification scores are given according to the homology distance to obtain the final identification results.

Types of MALDI-TOF



The principle of this method is to co-crystallize the sample with a mixture of small molecule matrix (cinnamic acid, mustard acid, and their articulates). When the UV laser (337 nm) irradiates the crystal, the matrix molecules absorb energy to desorb and ionize the sample with the matrix molecules. The ions generated from the sample acquire the same kinetic energy under the action of an accelerating electric field and pass through a vacuum electric field-free flight tube, the lighter ions are faster and reach the detector earlier, the heavier ions reach the detector later, and the flight time is proportional to (m/z). the ions generated by MALDI are mostly singly charged ions, and the spectral peaks in the mass spectra have a one-to-one correspondence with the mass number of each component of the sample, therefore MALD-MS is most suitable for the analysis of peptide mixtures after protein hydrolysis. the mass spectra of MALDI are cumulative of the results of multiple scans, making this instrument the most sensitive mass spectrometer available today, suitable for the analysis of trace samples (fmol-amol).

Electrospray ionization mass spectrometry (ESIMS)

The electrospray ionization method uses a high voltage to charge the droplets coming out of the capillary column at the inlet end of the mass spectrometer, and under the action of a reverse N2 gas stream, the droplets evaporate the solvent, the surface shrinks, the surface charge density increases, and the droplets burst into charged sub-droplets. The voltage on the surface of the droplet is so strong that the analyte ionizes and enters the mass analyzer as single or multi-charged ions. The mass spectral peaks show a series of mass-to-charge peaks for this compound with different charges, which can be converted to singly charged molecular ion peaks by software calculations. This technique allows the analysis of samples in the fmolamol range.

Tandem mass spectrometry (MS/MS)

Tandem mass spectrometry allows selective analysis of a component of a mixture without separating the groups. The parent ions of the molecular ion peaks of all components are obtained by the first stage mass analyzer, and the parent ions for further analysis are picked out by a mass overrunner.

Collision induced dissociation (CID)

The fragment ions (daughter ions) are generated, and the daughter ions are fed into the second stage mass analyzer to obtain the fragment peaks of the parent ions. By studying the cleavage relationship between the parent and daughter ions, structural information of peptides and proteins can be obtained.

Electrospray quadrupole time-of-flight tandem mass spectrometer (ESI-QTOF)

It is a new type of biological mass spectrometer introduced in recent years. Its primary mass spectrometer is an electrospray ionization source with a quadrupole mass analyzer, and the second mass spectrometer is replaced by a time-of-flight mass analyzer, which improves the resolution and sensitivity of the instrument and allows serial analysis of trace samples.

How to use MALDI-TOF?

Pre-specimen preparation
MALDI-TOF MS mass spectrometers are currently not adapted to mixed cultures, which must be fractionally pure microorganisms. The choice of culture medium, solid pellet, and liquid medium theoretically differs somewhat and is generally unaffected in practice. A solid medium is recommended for routine identification.
For solid media, theoretically selective and non-selective media will not have much effect on mass spectrometry identification, but it is still recommended to try to choose non-selective media for bacterial culture. 105-107 colonies (CFU) are usually required for MALDI-TOF MS identification to form the number of bacteria. The culture time as follows needs to be noted: a short period of culture generally does not affect the identification results, but because it is impossible to determine whether the pure colonies, such as mixed growth may affect the identification results failure. For spore-producing microorganisms, the too long incubation time will lead to a decrease in protein peak signal and plot quality, such cases need to be identified after passaging.

Target painting
Microbial colonies are applied to the central location of the target and spread evenly to form a thin layer.
When applying the target, it is necessary to pay attention to the following: too little amount of bacteria is applied, resulting in insufficient analysis signal and leading to failure of identification; too much bacteria is applied or unevenly applied, which can also lead to failure of identification. When coating should try to cover the whole target area, beyond the target area is easy to cause cross-contamination.
For mucoid colonies: because the identification system mainly detects intracellular ribosomal proteins, mucoid bacteria due to the formation of extracellular mucopolysaccharide pods dilute the concentration of bacteria coated onto the target site, which may lead to identification failure. For mucoid bacteria, the following methods can be used to enhance the identification: ① Mix the mucoid bacterial colonies well with the inoculation loop, and then pick some samples for spotting; ② Gently brush off the mucus on the surface of the colonies with a cotton swab, and then pick some lower samples for spotting; ③ Pick the fractionated colonies with the inoculation loop into EP tubes with 300 μl of deionized water, fully disperse and wash, 16 000~19 000× g, centrifuge for 2 min, discard the supernatant, and take the precipitate to spot plate.
For dry-type colonies: care should be taken to avoid picking the medium components, as far as possible in the early stage of colony growth is more sparse picking, can try to use formic acid in the test tube for extraction before coating the target plate.

Matrix fluid
Add 2μLB liquid drop by drop with a pipette, let it dry naturally, then add 1.5μLA liquid drop by drop in the middle of the target, dry, and wait for measurement.

On-board testing
Follow the MALDI-TOF instrument operating instructions (including pre-batch calibration)

Collecting data and comparing it with database to give identification results
To ensure the reliability of clinical identification results, the identification results obtained by MALDI-TOF MS should be reviewed by microbiology professionals before reporting, and the accuracy of the results can be judged during the review process by combining the sample source, staining results, morphology, culture conditions and other characteristics, and secondary identification or additional auxiliary experiments should be performed for suspicious results, and if inconsistent results occur, they should be promptly investigated reasons and give reasonable explanations. Since the interpretation of the results of different MALDI-TOF MS systems is not the same, the following is an example of the reporting and interpretation of only two results that are more used in the market, for other MALDI-TOF MS systems, refer to the manufacturer's instructions.

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