The matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an analytical method of microbial identification and characterization based on the fast and precise assessment of the mass of molecules in a variable range of 100 Da to 100 KDa.

The first description of the use of MS technology for bacterial identification was in 1975 [1Anhalt JP, Fenselau C. Identification of bacteria using mass spectrometry. Anal Chem 1975; 47: 219-5.]. Furthermore, MALDI-TOF MS was used by the mid-1990s for the identification of bacteria in research settings [2Claydon MA, Davey SN, Edwards-Jones V, Gordon DB. The rapid identification of intact microorganisms using mass spectrometry. Nat Biotechnol 1996; 14: 1584-6., 3Holland RD, Wilkes JG, Rafii F. Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 1996; 10: 12227-32.] but it took a long time for the introduction of this technology in routine microbiology. Then, in 2004, the first complete database for bacterial identification was reported [4Keys CJ, Dare DJ, Sutton H. Compilation of a MALDI-TOF mass spectral database for the rapid screening and characterisation of bacteria implicated in human infectious diseases. Infect Genet Evol 2004; 4: 221-42.].

The possibility of MALDI-TOF to quickly characterize a wide variety of microorganisms including bacteria, fungi and viruses [5Giebel R, Worden C, Rust SM. Microbial fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) applications and challenges. Adv Appl Microbiol 2010; 71: 149-84.] increase it potential application in some areas of microbiological diagnosis. Thus, this technology can be used for rapid microbial identification at a relatively low cost and it is an alternative for conventional laboratory diagnosis and molecular identification systems. The major contribution of this method is that has drastically improved the time to identification of a positive sample.

The main use of this technology has been, until now, the identification or typing of bacteria from a positive culture. However, applications in the field of clinical virology have been also performed and are opening up new opportunities, although the introduction in the routine laboratory diagnosis still remains a challenge.

In this review, MALDI-TOF technique is presented including sample preparation as well as data management and quality control, mainly focused in viral research and diagnosis.

The mass spectrometer is composed of three main components: an ion source to ionize and transfer sample molecules ions into a gas phase, a mass analyser device that separate molecules depending to their mass and finally a detector to monitor all separated ions. There are several ionization methods such as chemical ionization, electrospray and MALDI. The choice of method basically depends to the objective of the MS analysis and the nature of the sample, although both MALDI and electrospray are techniques that permit ionization and vaporization of a big quantity of biomolecules [6Emonet S, Shah HN, Cherkaoui A, Schrenzel J. Application and use of various mass spectrometry methods in clinical microbiology. Clin Microbiol Infect 2010; 16: 1604-3.].

In MS technique [7van Belkum A, Welker M, Erhard M, Chatellier S. Biomedical mass spectrometry in today's and tomorrow's clinical microbiology laboratories. J Clin Microbiol 2012; 50: 1513-7.], samples are prepared by mixing several biomolecules and embedded in a matrix that will crystallize inside. The matrix is composed of small acid molecules that have a strong optical absorption in the range of the laser wavelength used, although the composition is different according to the type of laser used and the biomolecule to be analysed. The molecules are then both desorbed and ionized by charge transfer by absorbing the energy of a short laser pulse. The desorbed and ionized molecules are first accelerated in an electrical field and ejected through a metal flight tube and collide with a detector at the end of the flight tube. This cycle is repeated at a frequency of 50 or more hertz. Thus, biomolecules are separated according to their mass, and create a mass spectrum that is characterized by both the mass and the intensity of the ions. Finally, the result is a spectral signature (spike representation) that is then searched for in the appropriate database for the identification of the microorganism, comparing with values provides by the manufacturer of the database (Fig. 1).

Fig. (1) Principles of MALDI-TOF MS technique. The samples are first inserted and embedded in a matrix. A laser pulse is applied in the matrix and the molecules are then both desorbed and ionized by charge transfer. The molecules are accelerated in an electrical field passing through a flight tube and collide with a detector. Biomolecules are separated according to their mass. Finally, a mass spectrum is created which is compared with a database for the identification of the virus.

On the other hand, electrospray ionization mass spectrometry (ESI-MS) generally uses DNA amplicons that are dissolved in a solvent and then injected in a conductive capillary. Inside this capillary, high voltage is applied resulting in the emission of aerosols of charged drops of the sample. After this, the sample is sprayed through compartments with diminishing pressure, resulting in the formation of gas-phase multiple-charged analyte ions, which are finally detected by the spectrometer. ESI-MS technology permits high resolution analysing the basic composition of amplicons [8Ecker DJ, Sampath R, Massire C. Ibis T5000: a universal biosensor approach for microbiology. Nat Rev Microbiol 2008; 6: 553-8.].

There are also two available methods for typing using this technology. The first method is the MALDI-RE that uses the iSeq method [9Stanssens P, Zabeau M, Meersseman G. High-throughput MALDI-TOF discovery of genomic sequence polymorphisms. Genome Res 2004; 14: 126-33., 10Honisch C, Chen Y, Mortimer C. Automated comparative sequence analysis by base-specific cleavage and mass spectrometry for nucleic acid-based microbial typing. Proc Natl Acad Sci USA 2007; 104: 10649-54.]. In this technique, RNA strands are cleaved and the products of cleavage are analysed by mass spectrometry, and their spectra are compared with the spectra of the reference sequences. Initially, MALDI-RE was used mainly for the detection of single-nucleotide polymorphisms, but currently there are additional applications for this method [11Honisch C, Chen Y, Hillenkamp F. Comparative DNA sequence analysis and typing using mass spectrometry.In: Sha HN.Gharbia SE., eds. Mass spectrometry for microbial proteomics. Chichester Wiley 2010; 443-62.]. The second method for typing is based on the use of ESI technology to analyse specific DNA amplicons (PCR-ESI-MS) [12Ecker DJ, Massire C, Blyn LB. Molecular genotyping of microbes by multilocus PCR and mass spectrometry: a new tool for hospital infection control and public health surveillance. Methods Mol Biol 2009; 551: 71-87.].

Other potential application of MALDI-TOF MS technology is to provide critical epidemiological data about viral infections. From an epidemiological point of view, one of the main applications of these methods is determining specific data of clinical isolates for viral diseases’ outbreaks. Because of their complexity, some viral typing techniques require specialized test methods and additional diagnostic devices. For this reason, the typing is not routinely performed in every laboratory, so these samples are being sent out of the hospital and this fact could delay the investigation of these outbreaks and hospital-associated infections. The introduction of MALDI-TOF MS technology in the laboratories can facilitate the performing of tests leading to improve the information to the infection control staff. The introduction of MS techniques into the clinical laboratory can provide accurate and rapid epidemiological data and it could have a critical role in hospital infection control and surveillance of viral outbreaks.

Table 1 summarizes the main applications of MALDI-TOF MS to clinical virology.

Table 1.

Main Applications of MALDI-TOF Techniques to Clinical Virology

Table 2.

Advantages and Disadvantages of MALDI-TOF MS Technology for Viral Analysis Compared to Other Methods

With regard to viral diagnosis, there are few studies comparing MALDI-TOF MS technology with other conventional based methods (e.g. viral, culture, nucleic acid based techniques). MALDI-TOF method successfully detects viruses in a wide variety of biological specimens, and the concordance rate between MALDI-TOF and these techniques is high. PCR based identification strategies have some limitations such as much longer turnaround time than MS, reagent and labour costs and some technical problems. On the other hand, MALDI-TOF technology can also be used to identify viruses in co-infected samples. These methods are capable of simultaneous detection of multiple pathogens in a single assay and can then prevent misdiagnosis and delays in treatment without increasing costs or adding a new step to the process.

With regard to the technical problems, PCR methods could have inhibitory particles and issues of contamination; thus, separate areas for sample preparation, amplification and analysis are needed. Furthermore, nucleic acid based techniques are expensive and time-consuming and appear in most cases less convenient than MS for routine laboratory identifications. For MALDI-TOF there must be significant progress made in closing the upfront of PCR processing to include DNA extraction and the PCR process itself.

MALDI-TOF MS methods also allow large-scale research studies not only for fresh samples but also on archival samples from several biological specimens.

The main advantages and disadvantages of MALDI-TOF MS technology are shown in Table 2.

The most remarkable differences between MALDI-TOF method and other conventional techniques are the estimated time and costs required for sample identification. There are no studies of cost-effectiveness in viruses, but it is estimated that the cost of bacterial identification represent only 17-32% of the costs of conventional identification methods [32Seng P, Drancourt M, Gouriet F. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis 2009; 49: 543-1.]. Other studies have shown that the cost of reagents required for phenotypic and nucleic acid identification is higher than for MS techniques [33Cherkaoui A, Hibbs J, Emonet S. Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level. J Clin Microbiol 2010; 48: 1169-75.]. The expensive cost of MALDI-TOF devices is comparable with other laboratory equipment. Moreover, MALDI-TOF is a rapid technique if compared with other methods, so this technology improves the working time (e.g. pre-analytical procedure) as well as the turnaround time (e.g. analytical proceduce). The time needed for microorganism identification is < 15 minutes for MS method, whereas conventional techniques need 5-48 hours.

One of the main weaknesses of MALDI-TOF is that the identification is limited by database. The mass profile is used as a mass spectrum to compare with those of well-characterized organisms in a database. The spectrum typically includes specific peaks, so that with a comprehensive library of spectra, the identification might be performed by using bioinformatics.

Currently, there are a wide number of references in the database from the available commercial platforms for MALDI-TOF techniques. These mass spectra can establish the comparison and further identification of microorganisms, but until now these spectra are limited. The database will expand due to the collaboration between commercial companies and the hospitals of several countries, so the available databases should be improved in the near future. It will be very important the comparison of data from different companies’ instruments, but this fact is currently not feasible.

Sample preparation by MALDI-TOF MS remains laborious today, although some robots are being developed in order to improve the deposit of the sample and the matrix on the target. Moreover, some aspects of MALDI-TOF MS technology are being also investigated; for example, the laser pulse frequency, disposable target slides and the mass spectrometers as well as the software used for the communication between laboratory equipment and laboratory information systems.

Data management primarily depends on the interpretation of crude data. Currently, some methods for data interpretation have been investigated [34Welker M. Proteomics for routine identification of microorganisms. Proteomics 2011; 11: 3143-53.]. An important fact is the integration of information communication systems to physicians that requires hospital or laboratory information systems (HIS, LIS).

Reference databases require updating if new information is obtained. The addition of new isolates or mutations could improve this tool and the final diagnosis of the diseases. A robust system of information for regional prevalence of infectious diseases, emerging and re-emerging infectious pathogens, the discovery of new mutations and local or pandemic changes in epidemiology is necessary.

The MALDI-TOF MS system is increasingly used in clinical diagnostic laboratory for viral identification, mutation analysis, genotyping and antiviral resistance studies. However, there are still some problems arising during sample extraction affecting to the extraction protocol and to the adequate reagent conservation for the assay. These problems could be avoided by the introduction of an adequate quality control program. The extraction step should be daily checked by training technicians as well as the introduction of routinely internal quality controls.

Other aspect of this technique that must be submitted to an adequate quality control is the deposit of the sample on the microplate and the cleaning of this microplate. It is necessary implement a control about these issues in order to the correct identification of the samples.

Finally, it is a good practice for these devices the adequate maintenance of the MALDI-TOF based on a global quality control program. In this sense, the calibration control is a tool useful to recalibrate the spectrometer device and to identify technical problems. Appropriate maintenance of these devices is essential to carry out an adequate and accurate viral identification.

It is essential both to continuously improve the quality of the database and to introduce a quality control program for the MS device. The implementation of these programs can help to improve the quality of the overall procedure, so it will be the basis to improve the usefulness and the accuracy of MALDI-TOF.

MALDI-TOF MS is a relatively recent innovation for the identification of microorganisms and is being used in many clinical microbiology laboratories. The main application of this technology is, at the moment, the identification of bacteria to species level in less than a minute, although this method is about to become the standard of identification of microorganisms in most laboratories.

MALDI-TOF MS technology is also being introduced in virology assays. The main applications in this field are the identification of viruses from clinical samples, the discovery of mutations for these viruses, the rapid screening of virus subtypes, its use in molecular epidemiology and the detection of resistance to several antiviral agents.

Future applications of MALDI-TOF MS in clinical virology will be developed in order to provide more accurate identification on clinical specimens and will include the detection of resistance protein markers and specific virulence for several viruses. However, the introduction of this technology routinely for virology diagnosis would enable a change in working practices. Moreover, this fact will require a complete integration into laboratory workflow and several technical improvements such as to detection sensitivity. The continuous updating of databases for microorganisms will be also an important issue, as well as the possibility for the diagnosis directly from clinical samples.

MS methods can be used currently for viral diagnosis but refinement of technology will be an important approach for the introduction of MALDI-TOF in routine viral diagnosis.