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clinical settings. More rapid tech-niques have been introduced in rou-tine analysis. Serology (ELISA, im-munofluorescence-based detectionkits), molecular biology (e.g. nucleicacid hybridisation, PCR, sequencing),as well as other methods such as flowcytometry, are now saving identifica-tion time and costs. Each method,however, has advantages and disad-vantages and is often confined to theidentification of only selected organ-isms. At present none is fully satisfac-tory as far as speed, accuracy and pre-cision and total absence of drawbacksare concerned [1].Recently mass spectrometry (MS) hasbeen used for the identification of mi-croorganisms, mainly bacteria. Themethod, based on the characterisationof biomarker molecules, is rapid andaccurate and constitutes a valid alter-native to conventional methods ofidentification and classification in mi-crobiology [1, 2].

Mass spectrometry (MS)Mass spectrometry is an extremelysensitive technique that has beenknown for almost a century. Analysisof biomolecules by MS dates back tothe 60s, but its application was limitedby the fact that it could not be used todetect molecules of low volatility and

heavy weight (over 20–30 kDa) as wellas complex mixtures. These limita-tions were overcome in the late 80swith the introduction of new ionisa-tion techniques for non-volatile mole-cules, including proteins and peptides[1].MS is based on the ionisation of mole-cules. The formed ions run in a mag-netic or electric field and are then sep-arated and detected in a mass spec-trometer according to their mass-to-charge ratio (m/z). The spectra origi-nating from the analysis of complexmixtures, such as a bacterium colony,are characterised by several peaks,among which some are typical andrepresentative of the taxon to whichthe analysed microbe belongs (fig. 1).Identification of peaks by MS includetransfer of the analyte in the gas phase,ionisation (and possibly fragmenta-tion) of the molecules, acceleration ofcharged ions, separation of ions ac-cording to their m/z, detection of ionsand interpretation of the mass spec-trum obtained. The peak pattern isthen used to characterise and eventu-ally to identify the microorganismsstudied.Various devices are marketed for thistype of analysis and, depending on thetechnique used, different mass spectraare obtained.

Mauro Tonolla, Cinzia Benagli, Sophie DeRespinis, Valeria Gaia, Orlando PetriniIstituto cantonale di Microbiologia,Bellinzona, Switzerland

Mass spectrometry (MS) was devel-oped at the beginning of the 20th cen-tury and is generally used to acquireknowledge of molecular structurebased on a mass spectral pattern con-sisting of a number of structurally re-lated mass spectral peaks. Since thefirst report showing the ability of MSto detect biomarkers unique for se-lected microorganisms, knowledgeand the volume of reports on this topichave dramatically increased, espe-cially in the last five years. MALDI-TOF MS (matrix assisted laser desorp-tion ionisation – time of flight massspectrometry) is now increasinglyused in the diagnostic laboratory as acost effective, rapid and reliable tech-nique for the identification and typingof microorganisms. This article aimsto provide an overview of this technol-ogy, which was to have a major impacton microorganism diagnostics.

IntroductionMicrobiologists working in the diag-nostic laboratory have always strivento identify microorganisms as quicklyand accurately as possible as to genus,species and possibly the intraspecificlevel (subspecies, serogroup, genotypeor other subtypes).The classical methods in use so far in-clude the establishment of a pure cul-ture from which the germ can be char-acterised and identified using mor-phological, biochemical and chemo-taxonomic characters. Pure cultures,however, are time-consuming andoften difficult to obtain, in particularwhen dealing with fastidious organ-isms, e.g. intracellular pathogenic bac-teria.Rapid identification of microorgan-isms is important in several areas ofmicrobiology, but may be crucial in

Mass spectrometryin the diagnostic laboratory

Figure 1MS spectrum of an Acinetobacter calcoaceticus var. lwoffii strain.

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The mass spectrometerA mass spectrometer is composed offive main parts (Fig. 2):– A system for the introduction or in-

jection of micro- or nanograms of asolid, liquid or gaseous sample in themass spectrometer.

– The source of ions by which thesample is irradiated. Electron im-pact (EI), chemical ionisation (CI),fast atom bombardment (FAB),plasma desorption (PD), electro-spray (ESI), atmospheric pressurechemical ionisation (APCI), and ma-trix-assisted laser desorption/ionisa-tion (MALDI) are available; the mostappropriate is chosen depending onthe nature of the sample and the typeof analysis envisaged. ESI andMALDI can result in vaporisation ofbiomolecules with high molecularmass by transforming them into thegas phase from a liquid (ESI) or solidphase (MALDI).

– An analyser that separates ions ac-cording to m/z by applying an elec-tric or magnetic field. Magnetic field,time of flight (TOF), quadrupole (Q),ion trap (IT), and cyclotron reso-nance ion (ICR) are the most com-mon analysers used in MS.

– A sensor that detects separated ionsand amplifies the originated signal(gain sensitivity up to about 106).

– A computer for data processing ableto transform the information re-ceived by the detector to a mass spec-trum.

The performance of a mass spectrom-eter is characterised by:– The mass range, recognised as the

ratio m/z value.– The resolving power (R), which de-

notes the ability of an analyser todistinguish adjacent fragments. Fortwo ions neighbouring masses, Mand M + M, R = M / M. MS devicesmay have low (R <300), average (300>R >10 000) and high (R >10000) res-olution. If the resolution is highenough, it is possible to determinethe exact mass of each individualion.

– The scanning speed, the number ofscans per second required to obtaina full spectrum.

We shall now describe in more detailthe matrix-assisted laser desorptionionisation scanner and time of flight(MALDI-TOF) in view of its usefulnessin microbiological analysis.

Ionisation by matrix-assisted laserdesorptionThe classic ion sources use sampleevaporation by heating and ionisationthrough a high energy beam of elec-trons (approx. 70 eV). This techniquemay lead to a breakdown of the ana-lyte, which is of course undesirable forbiological molecules. For this reason,«soft» methods for evaporation and ir-radiation of the sample have been de-veloped. In fact, the application ofmass spectrometry to fragile biomole-cules has been the basis of part of thework that enabled Koichi Tanaka towin the Nobel Award for Chemistry in2002.MALDI meets this requirement byspraying (desorbing) and ionisingsample molecules through non-de-structive mechanisms which limitfragmentation. The two processes takeplace at the same time.Sample preparation for MALDI is car-ried out by mixing the sample with amatrix present in large excess (ma-trix/sample ratio: about 10000: 1). Thecomposition of the matrix variesdepending on the analyte and the typeof laser used. It is usually a weak or-ganic, non-volatile acid, with a chro-mophore absorbing light at the samewavelength of the laser used. The fivemost frequently used compounds aresinapinic acid, 2,5-dihydroxybenzoic

acid, 2,4-hydroxyphenyl benzoic acid(DHB), a-cyano-4-hydroxycinnamicacid and ferulic acid [7] (Lay 2001).The mixture is usually deposited on ametal support called target. After crys-tallisation of matrix and compound,the target is introduced into the ioni-sation spectrometer. The surface of thetarget is then bombarded with high-energy photons from a pulsed laserbeam, normally a nitrogen laser withan emission wavelength of 337 nm.The laser energy is absorbed primarilyby the matrix present on the surface ofthe target plate. This leads to the de-sorption of the analyte into thegaseous state with minimal warming.This process causes vaporisation ofboth ionic and neutral species (Fig. 3).Polar compounds such as proteins at-tract protons from the matrix, which isa weak acid and becomes chargedpositively. This may take place both inthe solid and in the gaseous phaseabove the target desorption surface. Inboth cases an analyte-proton gas phaseis formed. Neutral species are notdetected by the spectrometer andcharged matrix particles are ignoredbecause of the very different m/z ascompared with that of the sample.

MALDI mass spectrometry (Fig. 4) canbe used to detect non-volatile and ther-mally unstable molecules from a few toseveral hundred kDa; the usual inter-val is 2 to 20 kDa [2, 3, 4].This technique, usually coupled with aTOF (time of flight) analyser, is usednot only for the study of peptides andproteins, but also to analyse polymersugars or nucleic acids. In particular,proteins are an ideal target for analy-sis because they are good acceptors of

Figure 2Mass spectrometer: componentsand workflow (modified after [3]).

Figure 3The MALDI-TOF process. Courtesyof M. Welker, Anagnostec GmbH,modified.

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protons and have masses much higherthan those of the protonated matrixcompounds. The technology also pro-duces a single component in the spec-trum for each charged molecule. Frag-mentation may occur, but results inparticles of such magnitude that theycan easily be distinguished in the spec-trum. Hence each component in a mix-ture may be associated with a signifi-cant signal.MALDI has also been applied to theanalysis of proteins isolated from bac-terial extracts, including those that canbe used in the taxonomic classificationof bacteria. The great advantage of thismethod lies, however, in the possibilityof detecting proteins with taxonomi-cally relevant characteristics, and itsability to identify microorganisms di-rectly from whole cells without priorstages of separation, fractionation orwashing [1].

The time of flight analyser (TOF)The TOF analyser is often coupledwith a MALDI ionisation system(known as MALDI-TOF MS) for theanalysis of biomolecules.The device is simple and consists of ametal tube subjected to a vacuum. Ahigh voltage (ca. 20000 V during somems) is generated in the ionisationchamber and creates an electric fieldthat transmits the kinetic energy to theformed ions. The ions are accelerated,move from the target to the detectorand during this trip are separated ac-cording to their speed. Thus, the timespent to reach the detector (flight time)is dependent on the mass and chargeof each ion.Often the value of the charge is 1 andm/z corresponds to the numericalvalue of the mass. Small ions movefaster than larger ions.

The detector placed at a fixed distancefrom the source of ions allows correla-tion of the time employed by the ionsto reach it in relation to the m/z.A TOF analyser is characterised by afast scanning speed (flight times areusually in the order of milliseconds), alarge mass range (few kDa to over 300kDa) [1], which is essential for analy-sis of biomolecules with high molecu-lar weight and high sensitivity. Itsmain limitation is low resolutionpower, which is in the range of 300 to400 and does not distinguish betweenpeptides with masses differing by onlya few Da. The resolution of TOF sys-tems could be improved by introduc-ing reflectron systems (Fig. 5) makingit possible to reach resolutions close to5000, though this reduces the quantityof ions detected.

Identification of microorganisms byMALDI-TOF mass spectrometryThe versatility and speed of thismethod have rapidly conquered sev-eral fields related to the characterisa-tion of microorganisms. A Medlinesearch carried out using the keywords«microbiology taxonomy MALDI-TOF» has given 61 hits between 1998and 2008, of which 49 were publishedafter 2003. In fact this technique nowplays an important role in water, airand food quality monitoring, in med-ical and veterinary diagnostics, foren-sic investigations (including cases of

bioterrorism attacks), and environ-mental microbiology [1, 2, 3, 5 , 6].

PrincipleMALDI-TOF MS can be used for theidentification of bacteria, viruses,fungi and spores [2, 3, 7, 8, 9].Sample preparation is straightforwardand spares the researcher time-con-suming separation steps. The turn-around analysis time is very short (afew minutes), thanks inter alia to thepossibility of using whole, untreatedcells for the analysis. These are trans-ferred to support plates as suspensionand/or directly by picking the colonyfrom a solid medium. A small volume(e.g. 0.5 microlitres) of matrix solution(DHB or a-cyano-4-hydroxycinnamicacid) is then poured over the spottedmicroorganisms, thus causing the dis-ruption and death of cells and the for-mation of matrix crystals harbouringthe molecules needed for analysis. In-

Figure 4Model of a mass spectrometer.Courtesy of Shimadzu Ltd. Figure 5

Diagram of a TOF analyser: reflec-tron mode (high resolution and lowsensitivity for masses 995 kDa).

Figure 6General workflow for microorganism identification/classification.

ProcessingComparisonIdentification

Transfer of cellsfrom selectedcolonies

Release of

result(to LIMS)

Automated MALDI-TOFmeasurement

Spotting ontarget plates

N R . 3 | J U N I 2 0 0 9 THEME 23

tion are considered intact proteins, be-cause most prokaryotic proteins be-long in this interval [2], as shown inFig. 7.Prerequisites for correct identificationof microorganisms with MALDI-TOFare the use of type isolates and in-clusion of the results of their charac-terisation in a database consisting ei-ther of a library of spectra of knownsamples or of proteins against whichnew identifications can be compared(Fig. 8). The quality of the database tobe used to compare the spectrum ofthe unknown microorganisms is cru-cial. At present libraries of knownspectra that permit identification ofmore than 90% of clinically relevantbacteria are commercially available(e.g. SARAMISTM Anagnostec, http://www.anagnostec.eu/; Maldi BiotyperTM,Bruker, http://www.bdal.com/; Micro-beLynxTM, http://www.waters.com/). Inthe case of protein samples, the infor-mation accessible through genomicdatabases can be used, but these arenot standardised for taxonomic use[1].Identification by comparison of un-known to reference spectra involvesanalysis of the total spectrum andcomparison of only distinctive peaks.Comparing specific peaks is moreselective, because it “weighs” peaksspecific for a given microorganism, ex-cluding those originating from back-ground noise or exogenous factors.Comparing total spectra is also possi-ble and provides a fingerprint of thewhole reference organism. On theother hand, all peaks are used for theanalysis even if they are not specific forthe microorganism.For the definition of reference spectrait is important to use rigorous algo-rithms that take into account the ex-perimental variables affecting thespectra. The reproducibility of a spec-trum, which is primarily related to res-olution power, may be affected by dif-ferent factors such as growing condi-tions, growth media, the type of ma-trix, solvents and instruments (fig. 8)[7, 10, 11, 12].In general, organisms belonging to dif-ferent genera have very different spec-tra with few or no shared peaks. Con-versely, at the species and subgrouplevel, spectra are increasingly similar

and differences may be limited to onlya few (3–4) peaks [1, 13] that corre-spond to specific biomarkers for theorganism analysed and are often ex-pressed under all culture conditions,because in many cases they representhighly expressed housekeeping pro-teins such as ribosomal proteins [11,14]. On the other hand, the abundanceof signals is dependent on cultural con-ditions. Thus, the comparison andtherefore the characterisation of spec-tra must be carried out with softwarethat considers in particular the m/zratio rather than the abundance ofions [1].The specificity of MALDI-TOF identi-fication is very good. In our lab, 1021bacterial isolates from our routinediagnostic work were analysed usingthe Axima ConfidenceTM MALDI-TOF(Shimatzu Ltd.) coupled with theSaramisTM database (AnagnostecGmbH) and the identification resultswere compared to those obtained withthe biochemical testing systemPhoenixTM (Becton Dickinson).For 967 strains (95%) the results ofMALDI-TOF MS corresponded tothose obtained with the Phoenix sys-tem. In 63% of the discordant resultsthe MALDI-TOF MS identification wasconfirmed by additional biochemicalmethods (API) or PCR/sequencing.Thus, MALDI-TOF was able to identifycorrectly more than 98% of the isolatestested [15].

Taxonomic researchMALDI-TOF has been widely used toidentify and characterise microorga-nisms isolated from clinical and envi-ronmental samples.Adenovirus serotype 5, intact spores ofBacillus cereus and the yeast Saccharo-myces cerevisiae are among the organ-isms that have been studied and iden-tified using MALDI-TOF [2].Species in the fungal genera Serpula,Coniophora and Antrodia have alsobeen identified using MALDI-TOFspectra [16]. In our lab, MALDI-TOFMS has proven to be a powerful, reli-able, rapid and relatively inexpensivemethod of identifying Trichodermastrains at the species level. Furtherstudies are ongoing to evaluate theusefulness of the method for intraspe-cific characterisation [15].

activation with trifluoric acid prior tothe analysis is performed only forhighly pathogenic microorganisms asa precautionary biosafety measure [1,2, 3]. In general, colonies or a cen-trifuged portion of a liquid culture aredirectly used, while other MS tech-niques linked with chromatography orelectrophoresis call for prior treat-ments for separation of the compo-nents of interest [2].The resolution power of MALDI-TOFMS is very large since characterisationof whole cells involves analysis of avery complex mixture of molecules. Agood resolution is the starting pointfor obtaining reproducible and taxo-nomically valid spectral profiles [1].The sensitivity of the method is suchthat the detection limit lies around104–105 cells, depending on the speciesof microorganism investigated [3, 7].

Identification of microorganismsThe identification of microorganismsby MALDI-TOF mass spectrometry isbased on the detection of mass signalsfrom biomarkers that are specific atgenus, species or sub-group level [1].Protein biomarkers are the most char-acteristic and accessible molecules inthe analysis of whole cells, becausethey provide good signals without hav-ing to go through extraction, separa-tion or amplification steps. Moreoverthey constitute some 50% of the dryweight of vegetative bacterial cells,compared with 5–8% of polar lipidsand 0.01% of RNA. DNA, on the con-trary, is present only in one copy percell and is therefore inaccessible [2].A MALDI-TOF spectrum from wholebacterial cells typically contains 10–30peaks with masses ranging from 2 toabout 20 kDa. Their ions after desorp-

Figure 7Mass dependent distribution of pro-karyotic proteins (from the Swiss-Prot database).

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Slow growing, fastidious bacteria suchas Campylobacter coli, C. jejuni, andother species in this genus [18], as wellas some species of mycobacteria, havealso been successfully identified byMALDI-TOF analysis [19]. In 1999,Hathout and co-workers characterisedbiomarkers useful for the identifica-tion of spores of Bacillus species(B. subtilis, B. thuringensis, and B. an-thracis) [20]. Another group comparedthe phylogenetic trees of the generaMyxocococcus and Corallococcus ob-tained by MALDI-TOF MS and by se-quence analysis of the genes 16S rRNAand gyrB, and showed a high correla-tion between both methods [21].Other groups have focused their ef-forts on the development of standardprotocols, in particular to improve thereproducibility of the analyses. Repro-ducible and reliable profiles suitablefor the discrimination of 24 species ofbacterial pathogens and non-patho-genic Salmonella, Listeria and Es-cherichia were produced by Mazzeoand co-workers [22]. Salmonella andAcinetobacter were studied by Ruelle etal. [10], who developed a standardisedprotocol that has been applied to 65other bacterial species. Libraries of re-producible spectra have been obtainedfor several geno species of Aeromonasand Legionella [23, 24, 25], both of clin-ical and environmental origin. Arnold& Reilly [26] developed a mathemati-

cal approach with which to comparemass spectra and evaluate similarity,eliminating the subjective componentof visual analysis.The work of many research groups isnow directed towards the productionand collection of spectra from a varietyof species in different growing condi-tions [4, 12, 27, 28]. Valentine et al. [4]compared the spectra of three bacter-ial species (E. coli, B. subtilis and Y. en-terocolitica) grown on four differentculture media and observed variationsin spectra from one medium to an-other; they also observed the presenceof some peaks that can be consideredcharacteristic of the species and usedfor identification.Another factor affecting the spectra isthe age of the bacterial culture [7, 29].The authors reported that the spectraof cultures grown in the same condi-tions vary considerably for numberand intensity of the peaks dependingon the incubation time [29]. Parisi andco-workers [30] demonstrated cyclicaldaily protein expression for E. coli,whereas Welham et al. [8] highlightedchanges in spectra over time, with spe-cific peaks, however, persisting evenafter three months. Conversely, Bacil-lus atrophaeus did not show anychange in spectrum composition overtime [31].Wahl and co-workers [32] analysedmixtures of up to 9 bacterial species,

each with known spectra. All but onemixture had their bacterial compo-nents properly identified.Preliminary work indicates thatMALDI-TOF may also be useful in ge-netic research to detect PCR products[7, 33, 34]. Several studies suggest thatMALDI-TOF is a versatile tool for DNAand RNA fragment analysis after a pre-liminary PCR step [34–35]. Ikryan-nikova and coworkers [36] validated aMALDI-TOF MS minisequencingmethod for rapid detection of TEM-type beta-lactamases in Enterobacteri-aceae.

ConclusionsMALDI-TOF is a well-establishedmethod for direct characterisation ofbacteria, fungi, and even viruses. Sev-eral studies have shown that the re-sults of MALDI-TOF analysis are simi-lar to those obtained by phylogeneticanalysis (PCR, sequencing technolo-gies). In the diagnostic laboratory, theoutcome of MALDI-TOF identifica-tions is comparable to and as reliableas that originating from classic and es-tablished methods. The method is al-ready in use in public health laborato-ries (e.g. National Institute of Health,Gaithersburg, Maryland, and RobertKoch Institute, Berlin, Germany) toidentify prokaryotes of clinical impor-tance to genus, species and subgrouplevel and certifiy strains. In Europe, inMarch 2009, the AXIMA@SARAMIS™(Shimadzu Anagnostec) system foridentification of microorganisms ob-tained the official accreditation by theDACH (German Accreditation Coun-cil – Chemistry), responsible for theaccreditation of medical laboratoriesfollowing DIN EN ISO 15189 in Ger-many.MALDI-TOF is technically straightfor-ward, rapid and sensitive. It requiresminimal sample preparation and itscosts are low after a steep initial ac-quisition price for the spectrometer.Reproducible mass spectra are crucialfor the establishment of a validateddatabase, but the quality of the spectraneeds to be assessed depending on theinfluence of instrumental factors aswell as the growth conditions of themicroorganisms. Despite the variety ofMALDI-TOF methods proposed fortaxonomic analysis using whole cells,

Figure 8Result of a typical identification by comparison of spectra with SARA-MISTM. Green: perfectly matching unknown organism and reference;Yellow: identification with a wider error interval; White: correspondencelower than 80%.

N R . 3 | J U N I 2 0 0 9 THEME 25

velopment of standard protocols andsystems aimed at facilitating data in-terpretation, comparison of spectraand research databases.Unfortunately, speed of analysis iscounteracted by the need to grow cellsin culture media before analysis. Theemergence of new technologies andstrategies based on affinity capture ofspecific cells could make it possible toperform MALDI analysis with a

smaller number of cells, thus omittingthe preliminary stages of culture. Thisapproach would considerably increasethe already high potential of themethod.

Correspondence:PD Dr. Mauro TonollaInstitute of microbiology, Canton TessinLaboratory of microbial ecology, BioVeg UNI GEVia Mirasole 22A6500 Bellinzonamauro.tonolla@ti.ch

no standard conditions for the pro-duction of characteristic spectra haveyet been determined, and case-by-casevalidation is still needed before routineuse of this method in diagnostic labo-ratories.Current and future research musttherefore be directed towards investi-gating the factors influencing repro-ducibility, the detection of biomarkersexpressed in any condition and the de-

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