Matrix Assisted Laser Desorption Time Of Flight Mass Spectrometer (MALDI-TOF)

Background and History

Discuss about the Matrix Assisted Laser Desorption Time Of Flight Mass Spectrometer.

MALDI-TOF is an abbreviation for ‘Matrix Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometer, which is a mass spectrometry technology that allows the measurement of molecular mass of each atoms and compounds, converting them to charged ions, and can be used to analyze biomolecules (Ru.ac.za, 2018). The technique involves an ionization process called MALDI that utilizes a matrix that absorbs laser energy in a matrix to create ions, without fragmentation of a large molecule and mass spectrophotometric technique called TOF, which analyses the velocity of the created ions (recorded as the time of flight) as per the mass to charge ratio of the ion to differentiate ions of different masses and charges (Tuma, 2013).

The technique of matric assisted laser desoprption ionization (MALDI) was developed by two Deutsche scientists: Michael Karas and Franz Hillencamp in 1985. They found that alanine can be ionized more easily if mixed with tryptophan and then irradiated with 266nm pulse. The tryptophan was able to absorb the energy from the radiation and thereby ionize the non-absorbing alanine (Hillencamp & Karas, 2007). In 1987, Japanese engineer, Koichi Tanaka showed that large proteins like carboxypeptidase-A can be ionized by combining cobalt particles in glycerol and irradiating it with 337 nm nitrogen laser. This proved that in the right setup, large protein molecules can be ionized easily (Sekiya et al. 2005). The time of flight Mass Spectrometer was first used by A.E. Cameron and D.F. Eggers Jr. in 1948 (Katzenstein & Friedland, 1955; Mamyrin, 2001).

The following section will discuss the purpose and principles of MALDI TOF, followed by the procedure and application of the technique, discussion on typical resists obtained from MALDI TOF analysis, and also a brief discussing on the advantages and disadvantages of this technique.

Propose of this technique is the identification of bacterial isolates.

MALDI TOF allows a fast identification of clinical bacterial isolates using proteonomic based technique and protein profiling. This can be used as an alternative form of other identification techniques to recognize microorganisms like gram positive bacteria, Enterobacteriaceae, yeast, mold, non-fermenting bacteria and mycobacteria (Schulthess et al., 2013; Conway et al., 2011; Blättel et al., 2013; Lau et al., 2012; Degand et al., 2018; Panda et al., 2013).

The principle of this method is the identification of the plentiful proteins in the range of 2 to 20kDa by analyzing their mass (m) to charge (z) ratio (m/z value). This helps in the generation of a typical fingerprint for each type of microorganism, and can be used to compare to a reference spectra to identify the sample. The principle is based on the phenomenon of ionization of sample molecules when bombarded with laser.

Purpose and Principles

This provides a straightforward, simple and quick technique for sample identification, compared to genotypic and phenotypic processes (like immunological based techniques, fluorescent in situ hybridization, Microarrays, DNA sequencing, Loop mediated isothermal amplification, and metagenomic assay) (Panda et al., 2014). MALDI TOF allows spectrometry of large biomolecules like proteins, and peptides are converted to ions by the loss or addition of more than one proton. This is a ‘soft ionization’ process, which does not damage the structural integrity of the sample. The ions that are produced are then accelerated in a fixed potential which differentiates them on the basis of their mass to charge ratios. Various mass analyzers (like ion trap analyzer, quadrupole mass analyzer and time of flight analyzer) to detect and measure these charged analytes. The determination of, the mass to charge ratio is done  by finding put the time for the charged ion to travel through the length of the flight tube. The resultant information a peptide mass fingerprint (PMF) can be generated for the analytes present in a sample (Singhal et al., 2015).

The MALDI TOF comprises of 3 parts: ion source, mass analyzer and a detector. The MALDI (matrix) forms the source of the ion, while the flight tube and detector helps to detect and analyze the ions (ru.ac.za, 2018).

Vitek MS- This is an automated identification system for microbes that uses mass spectrometry technique and Matrix Assisted Desorption Ionization Time of Flight (MALDI-TOF) technologies. The instrument contains a comprehensive CE marked as well as database (for microbes) cleared by the FDA. The records include: accurate ID with Associated Spectra Classifier, integrated ID/AST result and allows complete flexibility and traceability (VITEK® MS, 2018).

Step 1: preparation of target slide introduction into a high vacuum chamber

Step 2: Sample is ionized using laser

Step 3: Protein cloud released and accelerated due to the electric field

Step 4: Time of flight of the protein is calculated

Step 5: Sensor detects the proteins to create a spectrum that shows the protein composition of the sample

(VITEK® MS, 2018).

Figure 1: Steps of Maldi Tof (source: Vitek® Ms, 2018)

• Zip-tip
• Acetonitrile (CAN)
• Trifluroacetic acid (TFA)
• Spotting matrix (alpha-Cyano-4-hydroxycinnamic acid)
• Calibration Mix
• Protein Sample

1. Equilibration: The Zip Tip is activated with 10 microlitre of acetonitrile (CAN) thrice.
2. Loading: The sample is loaded to the Zip Tip by pipetting the sample (5 to 10 microlitre at a time) repeated 15 times, and then the rest of the liquid is discarded.
3. Washing: Salts are removed by washing with 3×10 microlitre of 10% TFA the C18/C4 tip.
4. Elution: Sample is eluted from the Zip tip using 50% CAN in 0.1% TFA or directly into the matrix (like CHCA in 70% ACN/0.1% TFA).

The correct matrix is selected depending upon the molecular weight of the protein.

Sample	Matrix
Peptide less than 10 kDa	α-Cyano-4-hydroxycinnamic acid(CHCA)
Protein less than 10 kDa	a. Sinapinic acid     b. Super DHB
Polymer	a. α-Cyano-4-hydroxycinnamic acid(CHCA) b. 2,5 dihydroxybenzoic acid(DHB)
Glycosylated Protein	Super DHB

• The matrix solution is prepared in an a proper solvent [5 mg of alpha-cyano in total of 0.5 mL solution containing 0.2 mL of 0.1% TFA and 0.3 mL of 100% ACN]
• The standard solution or premix is prepared by adding 10 microlitre of each protein or peptide (10 picomoles per microlitre).

• 5 microlitre of the matrix solution is deposited into the spot plate and left for 10s, and the excess amount is then removed
• 5 microliotre of pepmix is added to the matrix solution and 0.5 microlitre of the matrix solution is then added to the sample (this is called the sandwich method). The step is repeated for other spots, so that each standard spot is surrounded with sample spots.
• The dish is kept in the drier for 30mins, until the spots are dry and have a uniform appearance (slightly yellow to off white color).

• 5 microlitre of matrix solution is added to the spot and left for 10 sec and the any remaining, additional solution  is removed
• 5 microlitre of the sample is added to the matrix solution and then 0.5 microlitre of the matrix is added back to the sample. The step is repeated for other samples, next to standard spot.
• The plate is kept in the drier, and the dried matrix ought to have a uniform look (slightly yellow to off white color).
• For further tracking, the spot positions (for standard and samples) are recorded.
• The plate is then inserted in the MALDI equipment (Iitb.vlab.co.in, 2018).

Applications: Bacteriology (detection of food and water borne bacteria, environmental bacteriology, detection and identification of bio-weapons, detecting and identifying antibiotic resistance in bacteria, bacterial strain typing and taxonomy), virology (clinical virology, viral genotyping and epidemiological studies), mycology (clinical mycology, detecting antibiotic resistance in fungi, fungal strain typing) (Singhal et al., 2015).

Procedure and Applications

 

Figure 2: MALDI TOF apparatus setup (source: Cobo, 2013)

Studies by Panda et al., (2014) that utilized MALDI TOF MS for the comparative analysis of 82 bacterial samples (and 12 ATCC controls), and compared to conventional techniques. The study showed that using MALDI TOF MS,, all the 12 ATCC reference strains could be properly identified with log values (score) of more than 2.30 (which means a high probability of correct identification). Comparison with the results found from MALDI TOF MS and conventional techniques showed discrepancy with 4 samples. These four samples were reanalyzed by a external laboratory, and three of them agreed with the MALDI TOF result, and therefore the accuracy of the MALDI TOF was calculated at 98.78% (81 samples of 82 identified correctly).

The figures below show the results of the study:

 

Figure 4: Confirmation of discrepancy in the results from MALDI TOF and conventional test; source (Panda et al., 2014).

It is fast, accurate, less expensive (than immunological based detection) and does not require trained personnel (Singhal et al., 2015). Wide variety of specimen can be characterized. The method is highly sensitive, providing high throughput and easy preparation of sample. The process can also be automated and helps to improve patient management (Cobo, 2013). It allows observation of ionized molecules without causing any fragmentation since the ions have low internal energy. It can help in the identification of microbes at the subspecies level (Augulis, 2018)

Initial cost of the equipment is high (Singhal et al. 2015). The process can also be time consuming, and can involve different genetic markers (Cobo, 2013). The sensitivity is low without a prior culture. The technique cannot detect a low amount of microbes in sterile samples (Augulis, 2018).

References:

Augulis, R. (2018). What are advantages and disadvantages of MALDI Imaging mass spectrometry vs next-generation sequencing in pathology research?. researchgate.net. Retrieved 3 March 2018, from https://www.researchgate.net/post/What_are_advantages_and_disadvantages_of_MALDI_Imaging_mass_spectrometry_vs_next-generation_sequencing_in_pathology_research

Blättel, V., Petri, A., Rabenstein, A., Kuever, J., & König, H. (2013). Differentiation of species of the genus Saccharomyces using biomolecular fingerprinting methods. Applied microbiology and biotechnology, 97(10), 4597-4606.

Cobo, F. (2013). Application of MALDI-TOF Mass Spectrometry in Clinical Virology: A Review. The Open Virology Journal, 7(1), 84-90. https://dx.doi.org/10.2174/1874357920130927003

Conway, G. C., Smole, S. C., Sarracino, D. A., Arbeit, R. D., & Leopold, P. E. (2011). Phyloproteomics: species identification of Enterobacteriaceae using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Journal of molecular microbiology and biotechnology, 3(1), 103-112.

Degand, N., Carbonnelle, E., Dauphin, B., Beretti, J. L., Le Bourgeois, M., Sermet-Gaudelus, I., … & Ferroni, A. (2018). Matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of nonfermenting gram-negative bacilli isolated from cystic fibrosis patients. Journal of clinical microbiology, 46(10), 3361-3367.

Hillenkamp, F., & Karas, M. (2007). The MALDI process and method (pp. 1-28). Wiley?VCH Verlag GmbH & Co. KGaA.

Katzenstein, H. S., & Friedland, S. S. (1955). New Time?of?Flight Mass Spectrometer. Review of Scientific Instruments, 26(4), 324-327.

Iitb.vlab.co.in. (2018). Experiment-5: Sample preparation for the MALDI-TOF MS analysis (Procedure) : Virtual Proteomics Laboratory : Biotechnology and Biomedical Engineering : IIT Bombay Virtual Lab. Iitb.vlab.co.in. Retrieved 3 March 2018, from https://iitb.vlab.co.in/?sub=41&brch=118&sim=414&cnt=2

Lau, A. F., Drake, S. K., Calhoun, L. B., Henderson, C. M., & Zelazny, A. M. (2012). Development of a Clinically Comprehensive Database and Simple Procedure for the Identification of Molds from Solid Media by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry. Journal of clinical microbiology, JCM-02852.

Mamyrin, B. A. (2001). Time-of-flight mass spectrometry (concepts, achievements, and prospects). International Journal of Mass Spectrometry, 206(3), 251-266.

Panda, A., Kurapati, S., Samantaray, J. C., Myneedu, V. P., Verma, A., Srinivasan, A., … & Singh, U. B. (2013). Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry. Indian journal of medical microbiology, 31(2), 117.

Panda, A., Kurapati, S., Samantaray, J., Srinivasan, A., & Khalil, S. (2014). MALDI-TOF mass spectrometry proteomic based identification of clinical bacterial isolates. PubMed Central (PMC). Retrieved 2 March 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4365351/

Ru.ac.za. (2018). Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometer. Ru.ac.za. Retrieved 2 March 2018, from https://www.ru.ac.za/media/rhodesuniversity/content/nanotechnology/documents/MALDI%20TOF%

Schulthess, B., Brodner, K., Bloemberg, G. V., Zbinden, R., Böttger, E. C., & Hombach, M. (2013). Identification of Gram-positive cocci by use of matrix-assisted laser desorption ionization–time of flight mass spectrometry: comparison of different preparation methods and implementation of a practical algorithm for routine diagnostics. Journal of clinical microbiology, 51(6), 1834-1840.

Sekiya, S., Wada, Y., & Tanaka, K. (2005). Derivatization for stabilizing sialic acids in MALDI-MS. Analytical chemistry, 77(15), 4962-4968.

 Tuma, R. (2013). MALDI-TOF Mass Spectrometry. Oncology Times, 25(19), 26. https://dx.doi.org/10.1097/01.cot.0000290986.00178.61

VITEK® MS. (2018). bioMérieux Clinical Diagnostics. Retrieved 3 April 2018, from https://www.biomerieux-diagnostics.com/vitekr-ms-0