Faculty of Pharmacy Microbiology Department
Contents What is MALDI-ToF MS? .................................................................................................... 3 Principle of MALDI-ToF MS ............................................................................................... 3 Matrix ................................................................................................................................ 3 Laser .................................................................................................................................. 4 Desorbed/Ionized particles detector .................................................................................. 4 Technique overview and procedure....................................................................................... 5 Application of MALDI-ToF MS ........................................................................................... 6 Assessment of MALDI-ToF MS ........................................................................................... 7 Advantages ........................................................................................................................ 7 Disadvantages .................................................................................................................... 7 Conclusion ............................................................................................................................. 7 References ............................................................................................................................. 9
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What is MALDI-ToF MS? It is a method which is used recently to detect and identify pathogens of different types; bacteria, viral & fungi, on the basis of mass spectrometry (MS). MS is an analytical technique that was primarily developed to detect chemical compounds based on their masses after their vaporization, fragmentation and ionization, and consequently, the charged particle masses are measured by their mass to charge ratio (m/z). To clarify, if a compound has an atomic mass equals 60 and charge equals 3, then, its m/z value will equal 10 (Arnold, 2010; Leber, 2016). Figure I Bruker© MALDI Biotyper
Clinically, the idea of using MS to identify bacteria by analysis of their protein dates back to 1970s, in which the era of chemo-taxonomy has begun. Many different techniques were developed based on MS such as ESI-ToF, SELDI-ToF, QMSs and MALDI-ToF, electron spray injection time-of-flight, surface-enhanced laser desorption ionization time-of-flight, quadrupole mass spectrometry and matrix-assisted laser desorption ionization time-of-flight, respectively. MALDI-ToF is considered the most costeffective method, thus it is the most commonly used, presently. At first, MALDI-ToF was used to detect pathogens based on prepared extracts of them but several protocols have been improved over years until a whole intact cell of bacteria can be identified now (Bauer and Popp, 2015).
Principle of MALDI-ToF MS MALDI technique is based on ionization and desorption of organisms’ protein to be identified, where protein extract or whole bacterial cell is deposited on a plate (target) on which laser emission pulses are applied. However, it wouldn’t work unless a matrix is added to preserve the tertiary structure of the protein from fragmentation power of the laser to detect it purely thus it is named Soft Laser Desorption (SLD) (Gharbia and Shah, 2017) (Arnold, 2010). Matrix The first introduced SLD matrix compound was a mixture of finely powdered metals and glycerol by Koichi Tanaka who shared a Nobel Prize in Chemistry in 2002 because of its discovery. After that, α-Cyano-4-hydroxycinnamic acid (CHCA) was introduced by Cain 3
and colleagues that was a point of turn as it eased detection of intact cells instead of protein extract. Consequently, several other matrices have been discovered and classified based on their application as follows (Arnold, 2010) (Bauer and Popp, 2015). Compound
Application
a-Cyano-4-hydroxycinnamic acid (CHCA)
Peptides <10 kDa (glycopeptides)
Sinapinic acid (3,5-dimethoxy-4-hydoxycinnamic acid) (SA)
Proteins >10 kDa
Super DHB, mixture of 10% 5-methoxysalycilic acid (2hydroxy-5-methoxybenzoic acid) with DHB
Proteins & glycosylated proteins Neutral carbohydrates & synthetic
2,5-Dihydroxybenzoic acid (DHB)
polymers
3-Hydroxypicolinic acid
Oligonucleotides
2-(4-hydroxy-phenlyazo)-Benzoic acid
Oligosaccharides & proteins
(HABA) Table 1 Describes several matrices and their applications.
Eventually, some other organic compounds may be added to the matrix to improve results by increasing accuracy and quality of spectrum to be produced. Acetonitrile or a mixture of ethanol\methanol in addition to strong acid; trifluoro acetic acid are typically used. Laser Laser emitted pulses are of duration up to few nanoseconds to vaporize and excite co-crystals of the matrix and the analyte into gaseous state. The laser used may be a neodymium/yttriumaluminum-garnet (Nd-YAG) laser at wavelength of 355 nm or nitrogen laser of 337 nm wavelength (Arnold, 2010). Desorbed/Ionized particles detector The ionized desorbed proteins are detected and measured using ToF method as follows. The ions enter the vacuum flight tube and accelerated by electrostatic field with the same kinetic
Figure II The principle of ToF detection
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energy at a fixed point and a fixed initial time. This lead to separation of ions based on their mass to charge ratios. The lighter ions reach detector first as they travel faster while the heavier ones reach last. Thus, time of flight of ions recorded is proportional to their mass, therefore, it is called Time-of-Flight detector (Arnold, 2010).
Figure III represnts the general principle of MALDI-ToF technique
Finally, the data result is plotted as mass spectrum on a computer. Each organism reveals a characteristic mass spectrum is compared the prepared database to identify the pathogen.
Technique overview and procedure There are specific protocols for each organism but, generally, the identification of organisms is done either by analysis of whole cell, specifically for major of bacteria and yeasts, or of protein extracts which is applied on fungi, and minority of bacteria and yeasts (Leber, 2016). â&#x20AC;˘
First step is extraction (for organisms that cannot undergo whole cell analysis), in which formic acid, acetonitrile or ethanol may be used to solubilize proteins then the sample is centrifuged at 10,000:13,000 rpm. Consequently, the supernatant is used in MALDI-ToF MS analysis.
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Second step is inoculation, either colonies isolates or an extract are applied directly on specific spots on the target slide followed by matrix application. Then, the prepared sample spots are dried in air.
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Finally, the samples are ready for the final process, identification, this process is based
Figure IV Represnts second step of inoculation of sample on target slide
on matching the resulted spectra to a reference database.
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Above all, the FDA database doesnâ&#x20AC;&#x2122;t have yet reference spectrums for all of organisms to be analyzed through MALDI-ToF such as aerobic actinomycetes and filamentous fungi (Leber, 2016).
Figure V Diagramatic workflow of MALDI-ToF MS analysis
Application of MALDI-ToF MS According to Sparkman and Watson (2013), it aids in several fields but with greater extent in biomedical sciences. First, the main application of it is identifying pathogens of different kinds including bacteria, viruses, fungal spores. Secondly, it is used extensively to detect protein structures; sequence of peptides and amino acids. It also helps in characterization of carbohydrates, glycoconjugates, protein-protein interactions as well as isozymes. Moreover, it aids in detection of some biomarkers such as analysis of saliva for oral cancer, diabetes screening based on quantitation of glutathionylated hemoglobin, and analysis of cryo-preserved section of murine brain tissue as well as trypsinized human T47D cells of breast cancer. In addition, it is used in quantitation of several biomolecules such as insulin, homovanilic acid, epinephrine, and growth hormone by the aid of specific matrices. Other analytical applications include synthetic polymers analysis such as polystyrene nanoparticles up to 1 MDa, copolymers of ethylene and propylene oxides and other several 6
polymers were analyzed using pencil lead as the matrix, and small molecules analysis such as pesticides. Finally, it is used to analyze tar pitch, creosote, polysulfonated azo dyestuffs, glycosphingolipids and samples of forensic interest (Sparkman and Watson, 2013).
Assessment of MALDI-ToF MS According to Bailey et al. (2013) and Carroll et al. (2012) Advantages •
It requires the least time to identify the pathogens.
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It can detect pathogens within blood and urine samples
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The best choice of detection method for fastidious
and
slow-growing
microorganism. •
Analysis using this method yield less wastes than standard protocols.
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Easy to use as there is no special skills are required by personnel.
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Chart 1 Represents time required to identify of some bacterial species following standard protocol versus MALDI protocol
Shows a great potential for future detection of resistance mechanisms.
Disadvantages •
It is not suitable to find low number of bacteria that may be present in sterile sample such as CSF as its analytical sensitivity ranges between 105-106 bacteria per well.
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It may work as a potential for the lab workers to lose the skills of identifying organisms by traditional standard ways.
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Further improvement is needed to differ between some specific species within the same genus.
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Very expensive instrument.
Conclusion MALDI-ToF MS is the most promising mass spectroscopy method for clinical microbiology. It is based on chemo-taxonomy of pathogens based on their proteins. The instrument itself is consists of laser (essential for ionization and desorption of the analyte), 7
slide target (upon which the sample is placed and air-dried before exposure to laser pulses) and Time-of-Flight detector (used to measure m/z value of ions produced according to their travel time within the detector). Matrix compound is applied to analytes prior to detection to save tertiary structure of protein from fragmentation, intense mass spectra and consequently improve the result. Its time efficiency will save lives specially for septicemic patient. Eventually, it is not only used for biomedical sciences but used in other fields to detect large mass molecules, specifically.
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References •
Arnold, E. (2010) Principles and Techniques of Biochemistry and Molecular Biology, 7th edition, Cambridge: Cambridge University Press.
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Bailey, D., Diamandis, E., Greub, G. and Poutanen, S. (2013) 'Use of MALDI-TOF for Diagnosis of Microbial Infections', Clinical Chemistry, vol. 59, no. 10, September, pp. 1435-1441.
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Bauer, M. and Popp, J. (2015) Modern Techniques for Pathogen Detection, Weinheim: Wiley Blackwell.
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Carroll, K., Ellis, B., Lee, R., Stamper, P., Tan, K. and Zhang, Z. (2012) 'Prospective Evaluation of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System in a Hospital Clinical Microbiology Laboratory for Identification of Bacteria and Yeasts', Journal of Clinical Microbiology, vol. 50, no. 10, October, pp. 3301-3308.
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Gharbia, S. and Shah, H. (2017) MALDI‐TOF and Tandem MS for Clinical Microbiology, 1st edition, Wiley.
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Leber, A.L. (2016) 'Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) for Microorganism Identification ', in Leber, A.L. Clinical Microbiology Procedures Handbook, 4th edition, ASM Press.
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Sparkman, D. and Watson, J. (2013) Introduction to Mass Spectrometry: Instrumentation, Applications, and Strategies for Data Interpretation, 4th edition, John Wiley & Sons.
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