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The essence on mass spectrometry based microbial diagnostics
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The essence on mass spectrometry based microbial diagnostics Magdalena Kliem and Sascha Sauer In recent years, matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry has become an important bioanalytical method to detect profiles of proteins and peptides derived from whole bacterial cells. This accurate molecular-phenotypic method can be easily applied to robustly detect bacteria on the genus, species and in some cases on the subspecies level. Standardised laboratory protocols for the preparation of abundant bacterial proteins and the development of tailored data analysis software, as well as highquality databases of microbial reference mass spectra, made the procedure attractive to replace phenotypic or biochemical procedures for identification of bacteria and other microorganisms. Moreover, genotypic and functional mass spectrometry based methods to detect for example bacterial strains or antibiotic resistance may become useful in the coming years. In general, mass spectrometry is a powerful tool to facilitate routine microbial diagnostics.
Address Max Planck Institute for Molecular Genetics, Otto-Warburg Laboratory, Ihnestrasse 63-73, D-14195 Berlin, Germany Corresponding author: Sauer, Sascha ([email protected])
Current Opinion in Microbiology 2012, 15:397–402 This review comes from a themed issue on Microbial proteomics Edited by Bertrand Se´raphin and Robert Hettich For a complete overview see the Issue and the Editorial Available online 10th March 2012 1369-5274/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2012.02.006
Introduction The best method for identifying microorganisms should provide universal, flexible, and quantitative identification capability even in complex samples. Further requirements for the wide acceptance of a diagnostic methodology include the accuracy and speed of analysis, the potential for high-throughput, and the low operational costs. Moreover, transfer, sharing, and storage of microbial data shall be made as simple as possible [1]. For decades a broad range of methods have been applied for taxonomic classification and identification of microorganisms [2], including phenotypic or laborious and expensive biochemical procedures such as Analytical Profile Index (API) analysis [3]. Currently, mass spectrometry revolutionises microbial diagnostics as this www.sciencedirect.com
method matches a number of key requirements for efficient bacterial diagnostics (Table 1). The principle idea of identifying microorganisms by mass spectrometry analysis of proteins stems from the 1970s [4]. The ongoing mass spectrometry based paradigm change in microbiology has mainly been driven by the validation and optimisation of robust standardised laboratory procedures since the late 1990s. Furthermore the availability of software packages for data analysis and the development of comprehensive databases of reference mass spectra of bacteria and other microorganisms were important to make mass spectrometry based bacterial identification applicable for daily practice. Companies such as Bruker Daltonics (http://www.bdal.com) and AnagnosTec (http://www.anagnostec.eu/), which made the application of mass spectrometry increasingly popular in many microbiology laboratories, were the key players in this area during recent years.
Mass spectrometry based protein mass pattern detection Compared to many other analytical tools using labelling, mass spectrometry is unique in a way that it detects an intrinsic physical property, the mass-to-charge (m/z) ratio of an analyte, leading to robust and precise analyses [5]. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) has been used to readily analyse peptides and to characterise proteins [6]. Electrospray ionisation mass spectrometry (ESI-MS) is another popular tool that detects these biomolecules, in which analytes are dissolved in organic-aqueous liquids [7]. In MALDI analytes are embedded in a crystal of small, acidic molecules termed ‘matrix’ such as alpha-cyano-4-hydroxycinnamic acid, sinapinic acid, or 2,5-dihydroxybenzoic acid (Figure 1). The analytes and the MALDI matrix are prepared on a conductive metallic ‘target’ plate that can be automatically introduced into the mass spectrometer. One spot on the target can be measured in a few seconds, which enables high-throughput detection. A MALDI mass spectrometer can detect efficiently various molecules of different masses simultaneously. Therefore MALDI mass spectra of protein fragments derived from bacterial samples can be used as fingerprints for rapid and reliable identification of bacteria (Figure 1). Similar molecular-phenotypic mass spectrometry based approaches are currently also being applied in clinical diagnostics of human patient body fluids to differentiate complex common diseases [8]. Protein mass pattern detection neither implies peptide sequence Current Opinion in Microbiology 2012, 15:397–402
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Table 1 Compilation of properties of MALDI mass spectrometry detection of protein profiles derived from bacterial cells Diagnostic feature
MALDI mass spectrometry detection of proteins derived from whole bacterial cells
Universal detection Flexible detection Quantitative detection Detection sensitivity
Specificity Speed High-throughput Costs Data handling
Widely applicable for a broad range of bacteria, potentially also for a number of other microorganisms. Yes. The workflow can be optimised for various microbial species. Very difficult with mass signal patterns. Only possible using some informative mass peaks. Complex samples require culturing and/or enrichment. A bacterial colony grown on solid media produces in general very reliable mass spectra. Usually, processing of 106–107 bacterial cells generates excellent mass spectra. Using enrichment methods less cells may be sufficient for detection. The method is well suited for rapid distinction of bacterial genera and species. Subspecies or strains may be differentiated in some cases, in particular using informative mass peaks. After culturing or enrichment, only a few minutes are required for analysis. Yes, several hundred samples can be analysed per hour with one MALDI mass spectrometer. Pipetting robots, tracing systems and automated mass spectrometry analysis devices are available. Low. Consumables cost a few cent per analysis. A linear automatic MALDI mass spectrometer costs about 120 000 US-Dollar or Euro. Easy. Simple software tools are available that avoid to a large extent in-depth analysis of mass spectra.
Figure 1
Chemical treatment (Optional) Transfer of protein extracts Bacterial samples Direct transfer of bacterial colonies Sample preparation on MALDI target metal plate
Intensity
MALDI-TOF-MS
m/z MALDI mass spectrometry
Protein mass pattern detection Current Opinion in Microbiology
MALDI-TOF MS protein mass pattern detection method. Bacteria, grown under controlled conditions or enriched from biological material, are pretreated by simple chemicals or directly applied with MALDI matrix onto a metal target plate. The plate is introduced into a MALDI mass spectrometer. These steps and data acquisition and analysis can be automated. Highly abundant proteins or protein fragments are monitored simultaneously by mass spectrometry. The signals detected are generally in the range of about 1000–20 000 m/z (mass to charge ratio). As is shown, a typical mass spectrum provides a specific molecular-phenotypic bacterial trait, profile or fingerprint for identification. Usually, only positively charged peptides or proteins are detected. In general most of the analytes detected in this mass range by MALDI have a charge z of 1 (singly charged), or to a lesser degree a charge z of 2 (doubly charged). The figure items were taken from ScienceSlides SuiteTM 2007. Current Opinion in Microbiology 2012, 15:397–402
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information nor identification of proteins. Precise mass signals of a protein fragment or protein sequence data can, however, improve accuracy in identifying bacteria and in particular be used to gain additional information for detecting bacterial antibiotic resistance or virulence factors. These sophisticated analyses though widely used in discovery proteomics based on sequenced organisms [9], they are still inexpedient in practice of a routine diagnostic laboratory. Many of the mass signals detected from bacterial protein extracts are derived from conserved ribosomal or other abundant structural or regulatory house-keeping proteins [10]. Owing to the detection of integral and ubiquitous cellular proteins of fragments thereof, the method is sufficiently stable against environmental factors only if conditions are not changed significantly during sample preparation. Protein mass pattern spectra can be analysed for identification of bacteria on the genus, species, and potentially even on the subspecies level [11,12]. Although the current emphasis lies on bacterial detection, the approach described can potentially be extended for detection of other microorganisms such as mycobacteria [13], fungi [14], and yeast [15], while viral detection will remain difficult as viruses have for example relatively lower protein content in general.
Sample preparation Whole bacterial cells can be easily processed with minor amounts of work and involving low-cost consumables [18]. A number of methods have been developed to generate samples from a large range of different bacterial genera. Usually a single bacterial colony is sufficient for MALDITOF analysis. In many cases culturing of bacteria is required to obtain enough material. A broad range of (selective or non-selective) solid or liquid media known from conventional procedures including blood cultures can be applied to various microorganisms but may have to be optimised from case to case. Moreover, direct microbial identification from blood, urine other body fluid or stool samples can be performed without culturing using enrichment methods [19]. In practice, culturing can be advantageous to gain initial information on the samples by distinguishing phenotypic features such as morphology (cocci or bacilli), Gram staining, culture characteristics, or antibiotic susceptibility profiles. In general subculturing requires at least an additional day but can produce more reliable mass spectral data, as a result of increased amounts of bacterial samples and/or reduced complexity of polymicrobial cultures.
Bacterial identification by mass spectrometry is a simple process. Mass spectra of reference bacterial strains are stored as reference libraries. For identification, suitable algorithms are applied to compare mass spectral fingerprints derived from bacterial probes with reference spectra [16]. For example, by pattern-matching algorithms, peaks in experimental mass spectra (Figure 1) that match with reference spectra and vice versa are counted, and intensities of matched signals are correlated with each other. Similarity or other score values obtained from this analysis can be compared with experimental threshold values applied for reliable identification on the genus and the species level. For identification of closely related subspecies, more indepth analysis of mass signals can be applied in which additional values are assigned to particularly informative signals observed in the reference spectra of bacterial samples [16].
Differential mass spectra of the same bacteria can occur owing to dramatically varying culturing conditions or preparation methods. Initially, this has impeded the application of the method. But application of validated and well-controlled growth media and standardised sample preparation procedures enabled generation of reproducible mass spectra for routine bacterial identification [20,21]. In some refined protocols chemical pretreatment during protein extraction is used to prepare sufficient proteins and peptides for MALDI analysis. For inactivating highly pathogenic bacteria and spores, fast pre-treatment steps that use for example ethanol or trifluoro acetic acid (TFA) have been developed [22]. The most convenient ‘direct transfer’ method omits any pretreatment. In this method, a bacterial colony is smeared onto a MALDI target as a thin film and MALDI matrix solution is laid over the already dried bacterial samples. This way, using acidic organic MALDI matrix, proteins and peptides are extracted from bacteria via sample preparation on the target plate and made that accessible to efficient laser desorption/ionisation.
Furthermore, bacterial mass patterns can be used as phenotypic traits for classification using unsupervised hierarchical clustering or other approaches applied in taxonomy [17]. As described, MALDI mass spectrometry analysis of bacterial cells particularly detects abundant housekeeping proteins, especially ribosomal proteins. Thus, alternatively to sequence analysis of 16 S rDNA mass pattern data of these proteins can be used as taxonomic markers to generate dendrograms for visualisation: The more similar the mass spectra, the closer the relationships of the bacteria [1,18].
Standardisation of laboratory conditions is particularly important to maintain identification capability of subspecies with slightly different but informative mass patterns. A number of protocols have been optimised with regard to handling of (specific) microbial samples and fine-tuning of MALDI analysis parameters [18,23], as well as optimisation of protocols for handling of blood cultures to remove interfering blood cells and host proteins [24]. Furthermore, mass spectrometry based bacterial identification has been largely successfully validated for comparison with conventional automated bacterial identification techniques and
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could produce very convincing intralaboratory and interlaboratory reproducibility [25,26]. The ability to differentiate closely related bacteria by mass patterns might also be improved by increasing the number of peptides using in addition to protein extraction proteases such as trypsin to digest bacterial proteomes [27]. The resulting protein fragment patterns can be compared against reference mass spectra produced by protease digestion of bacterial reference protein extracts. Standardisation of this approach may be more challenging than simple protein extraction methods as the reproducibility of protease digestion of whole proteomes of bacterial cells can be undesirable in practice.
Software tools Mass spectra can be easily digitalised, which enables straightforward data storage and exchange. For microbial identification and taxonomic classification user-friendly commercially software packages such as the MALDI Biotyper provided by Bruker Daltonics (http://www.bdal.com) and Samaris developed by AnagnosTec (http://www. anagnostec.eu/) are currently mainly used in the community. Besides these two widely proven software packages, recent data analysis tools such as Andromas [28] provide algorithms for reliable identification and classification of protein mass patterns, as well as steadily up-to-date reference mass spectra of several thousands of bacterial and other microbial entries. Using these data analysis tools, researchers can also create and upload their own library of reference mass spectra [16,29].
Performance in practice MALDI-TOF mass spectrometry based bacterial identification has been validated during the past years in a broad range of bacteria and microorganisms. Depending on the bacterial genera or species, over 90% to close to 100% experimental reproducibility and identification success rates were reported [30]. The accuracy was achieved with turnover rates of less than an hour instead of about one or two days required for the conventional methods, and a significant shortening of technician working time for sample and data processing. Moreover, the cost for consumables is estimated to be reduced to a twentieth compared to biochemical methods, while the mass spectrometer used has slightly higher costs than automated systems for large-scale bacterial identification by API [31]. In general, API and MALDI-TOF MS of protein mass patterns produce similar results. In the case of human pathogens such as Bacteroides fragilis, mass spectrometry even outperformed conventional assays [32]. Validation experiments using in parallel the MALDI Biotyper (Bruker Daltonics) and the Samaris (AnagnosTec) software revealed slightly different results, mainly as a consequence of varying bacterial entries in the commercially available reference spectra databases [33– 35]. Adding reference spectra of microorganisms that are either under- or not at all represented and increasing the Current Opinion in Microbiology 2012, 15:397–402
number of available mass spectra per species will steadily improve the performance of the method [36]. Well-organised community efforts including large reference centres to produce publicly available databases of standardised bacterial reference spectra, may make the methodology more independent from the commercially available spectra libraries. More comprehensive highquality reference spectra may even increase identification accuracy and accelerate acceptance of the promising method. Extensive double-blind studies on a number of different microorganisms involving several laboratories will further validate mass spectrometry based microbial identification systems [37]. Consequently, current efforts of the aforementioned companies in the field focus in particular on certification of the method for diagnostic applications.
Mass spectrometry detection of microbial nucleic acids In contrast to the phenotypic detection of protein profiles described so far, genotypic methodologies such as DNA sequencing, tailored PCR-based or restriction-enzyme based methods can more easily provide sufficient resolution to identify bacterial strains, and can be used to detect efficiently viruses. Mass spectrometry can not only detect peptides and proteins but also nucleic acids. For example, bacteria can be identified to the species level by protein mass pattern detection, while using the same instrument more in-depth information, if necessary, can be gained by additional mass analysis of their nucleic acids. PCR amplification and MALDI-detection based SNP-genotyping [38–40], MALDI-based re-sequencing [41,42] or electrospray ionisation mass spectrometry detection of PCR products [43,44] are new alternatives for the analysis of nucleic-acids based microbial markers, which may allow efficient monitoring of microbial epidemics [45]. In contrast to protein profiles, mass spectrometry based sequence analysis of microbial genomes can in principle rapidly and accurately reveal known or emerging antibiotic resistance mediated by gene mutations, mobile genetic elements or virulence factors, and detect viruses. Owing to the specific amplification of targeted regions of nucleic acids by PCR, low levels of bacteria can be detected quantitatively, even in higher complexity samples. In general, purification of DNA from bacteria is required for efficient amplification as the reliability of amplification of nucleic acids from crude mixtures is low. PCR also implies that potentially falsepositive PCR products can interfere with the analysis because even single copies of bacterial DNAs can potentially lead to striking signals and misinterpretation.
Routine diagnostics Mass spectrometry based protein profiling can be readily applied as its workflow fits well with classical culturing procedures. Interestingly, short and simple morphological www.sciencedirect.com
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and phenotypic testing such as Gram staining or detection of bacterial cytochrome c oxidases and/or catalase activity can be combined with mass spectrometry to prevent major misidentifications. The sample preparation steps after culturing are easily applicable and can be performed in a few minutes using simple reagents or kits [46,47]. Commercial solutions for automated handling and tracking of bacterial samples are available tailored for mass spectrometry analysis of bacteria. As protein profiling is not challenging in terms of mass accuracy and signal resolution, simple high-throughput MALDI mass spectrometers with minor requirements on maintenance can be applied. Therefore, owing to the availability of easily applicable software tools, the degree of training required for a technician is relatively low.
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With the further development and certification of the protein mass pattern detection method, current and near future applications include mainly medical and veterinary diagnostics applications, monitoring of phytopathogens, and ensuring food safety and quality control as are required in pharmacology, cosmetics, drinking water purification, and industrial microbiology. In the immediate future, mass spectrometry will certainly improve routine microbial diagnostics.
Acknowledgements We would like to thank Drs. Markus Kostrzewa, Thomas Maier, and Jo¨rg Sauer for fruitful discussions, Anja Freiwald for support, and Dr. ChungTing Han for critically reading of the manuscript. Our work is funded by the German Ministry for Education and Research (BMBF, grant number 0315082), the European Union (FP7/2007-2013, under grant agreement n8 [HEALTH-F4-2008-201418] entitled READNA, and [FP7/2007-2011], under grant agreement n8 262055 (ESGI). We apologise to all those, whose work cannot be covered here owing to space limitations.
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38. Storm N, Darnhofer-Patel B, van den Boom D, Rodi CP: MALDITOF mass spectrometry-based SNP genotyping. Methods Mol Biol 2003, 212:241-262. 39. Sauer S, Reinhardt R, Lehrach H, Gut IG: Single-nucleotide polymorphisms: analysis by mass spectrometry. Nat Protoc 2006, 1:1761-1771. 40. Bouakaze C, Keyser C, Gonzalez A, Sougakoff W, Veziris N, Dabernat H, Jaulhac B, Ludes B: Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based single nucleotide polymorphism genotyping assay using iPLEX gold technology for identification of Mycobacterium tuberculosis complex species and lineages. J Clin Microbiol 2011, 49:3292-3299. 41. Honisch C, Chen Y, Mortimer C, Arnold C, Schmidt O, van den Boom D, Cantor CR, Shah HN, Gharbia SE: Automated comparative sequence analysis by base-specific cleavage and mass spectrometry for nucleic acid-based microbial typing. Proc Natl Acad Sci USA 2007, 104:10649-10654. A MALDI-based re-sequencing method for microbial detection. 42. Honisch C, Mosko M, Arnold C, Gharbia SE, Diel R, Niemann S: Replacing reverse line blot hybridization spoligotyping of the Mycobacterium tuberculosis complex. J Clin Microbiol 2010, 48:1520-1526. 43. Ecker DJ, Sampath R, Blyn LB, Eshoo MW, Ivy C, Ecker JA, Libby B, Samant V, Sannes-Lowery KA, Melton RE et al.: Rapid identification and strain-typing of respiratory pathogens for epidemic surveillance. Proc Natl Acad Sci USA 2005, 102:8012-8017. 44. Sampath R, Russell KL, Massire C, Eshoo MW, Harpin V, Blyn LB, Melton R, Ivy C, Pennella T, Li F et al.: Global surveillance of emerging Influenza virus genotypes by mass spectrometry. PLoS ONE 2007, 2:e489. 45. Beres SB, Carroll RK, Shea PR, Sitkiewicz I, MartinezGutierrez JC, Low DE, McGeer A, Willey BM, Green K, Tyrrell GJ et al.: Molecular complexity of successive bacterial epidemics deconvoluted by comparative pathogenomics. Proc Natl Acad Sci USA 2010, 107:4371-4376. 46. Yan Y, He Y, Maier T, Quinn C, Shi G, Li H, Stratton CW, Kostrzewa M, Tang YW: Improved identification of yeast species directly from positive blood culture media by combining Sepsityper specimen processing and Microflex analysis with the matrix-assisted laser desorption ionization Biotyper system. J Clin Microbiol 2011, 49:2528-2532.
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Current Opinion in Microbiology 2012, 15:397–402
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The essence on mass spectrometry based microbial diagnostics Magdalena Kliem and Sascha Sauer In recent years, matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry has become an important bioanalytical method to detect profiles of proteins and peptides derived from whole bacterial cells. This accurate molecular-phenotypic method can be easily applied to robustly detect bacteria on the genus, species and in some cases on the subspecies level. Standardised laboratory protocols for the preparation of abundant bacterial proteins and the development of tailored data analysis software, as well as highquality databases of microbial reference mass spectra, made the procedure attractive to replace phenotypic or biochemical procedures for identification of bacteria and other microorganisms. Moreover, genotypic and functional mass spectrometry based methods to detect for example bacterial strains or antibiotic resistance may become useful in the coming years. In general, mass spectrometry is a powerful tool to facilitate routine microbial diagnostics.
Address Max Planck Institute for Molecular Genetics, Otto-Warburg Laboratory, Ihnestrasse 63-73, D-14195 Berlin, Germany Corresponding author: Sauer, Sascha ([email protected])
Current Opinion in Microbiology 2012, 15:397–402 This review comes from a themed issue on Microbial proteomics Edited by Bertrand Se´raphin and Robert Hettich For a complete overview see the Issue and the Editorial Available online 10th March 2012 1369-5274/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2012.02.006
Introduction The best method for identifying microorganisms should provide universal, flexible, and quantitative identification capability even in complex samples. Further requirements for the wide acceptance of a diagnostic methodology include the accuracy and speed of analysis, the potential for high-throughput, and the low operational costs. Moreover, transfer, sharing, and storage of microbial data shall be made as simple as possible [1]. For decades a broad range of methods have been applied for taxonomic classification and identification of microorganisms [2], including phenotypic or laborious and expensive biochemical procedures such as Analytical Profile Index (API) analysis [3]. Currently, mass spectrometry revolutionises microbial diagnostics as this www.sciencedirect.com
method matches a number of key requirements for efficient bacterial diagnostics (Table 1). The principle idea of identifying microorganisms by mass spectrometry analysis of proteins stems from the 1970s [4]. The ongoing mass spectrometry based paradigm change in microbiology has mainly been driven by the validation and optimisation of robust standardised laboratory procedures since the late 1990s. Furthermore the availability of software packages for data analysis and the development of comprehensive databases of reference mass spectra of bacteria and other microorganisms were important to make mass spectrometry based bacterial identification applicable for daily practice. Companies such as Bruker Daltonics (http://www.bdal.com) and AnagnosTec (http://www.anagnostec.eu/), which made the application of mass spectrometry increasingly popular in many microbiology laboratories, were the key players in this area during recent years.
Mass spectrometry based protein mass pattern detection Compared to many other analytical tools using labelling, mass spectrometry is unique in a way that it detects an intrinsic physical property, the mass-to-charge (m/z) ratio of an analyte, leading to robust and precise analyses [5]. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) has been used to readily analyse peptides and to characterise proteins [6]. Electrospray ionisation mass spectrometry (ESI-MS) is another popular tool that detects these biomolecules, in which analytes are dissolved in organic-aqueous liquids [7]. In MALDI analytes are embedded in a crystal of small, acidic molecules termed ‘matrix’ such as alpha-cyano-4-hydroxycinnamic acid, sinapinic acid, or 2,5-dihydroxybenzoic acid (Figure 1). The analytes and the MALDI matrix are prepared on a conductive metallic ‘target’ plate that can be automatically introduced into the mass spectrometer. One spot on the target can be measured in a few seconds, which enables high-throughput detection. A MALDI mass spectrometer can detect efficiently various molecules of different masses simultaneously. Therefore MALDI mass spectra of protein fragments derived from bacterial samples can be used as fingerprints for rapid and reliable identification of bacteria (Figure 1). Similar molecular-phenotypic mass spectrometry based approaches are currently also being applied in clinical diagnostics of human patient body fluids to differentiate complex common diseases [8]. Protein mass pattern detection neither implies peptide sequence Current Opinion in Microbiology 2012, 15:397–402
398 Microbial proteomics
Table 1 Compilation of properties of MALDI mass spectrometry detection of protein profiles derived from bacterial cells Diagnostic feature
MALDI mass spectrometry detection of proteins derived from whole bacterial cells
Universal detection Flexible detection Quantitative detection Detection sensitivity
Specificity Speed High-throughput Costs Data handling
Widely applicable for a broad range of bacteria, potentially also for a number of other microorganisms. Yes. The workflow can be optimised for various microbial species. Very difficult with mass signal patterns. Only possible using some informative mass peaks. Complex samples require culturing and/or enrichment. A bacterial colony grown on solid media produces in general very reliable mass spectra. Usually, processing of 106–107 bacterial cells generates excellent mass spectra. Using enrichment methods less cells may be sufficient for detection. The method is well suited for rapid distinction of bacterial genera and species. Subspecies or strains may be differentiated in some cases, in particular using informative mass peaks. After culturing or enrichment, only a few minutes are required for analysis. Yes, several hundred samples can be analysed per hour with one MALDI mass spectrometer. Pipetting robots, tracing systems and automated mass spectrometry analysis devices are available. Low. Consumables cost a few cent per analysis. A linear automatic MALDI mass spectrometer costs about 120 000 US-Dollar or Euro. Easy. Simple software tools are available that avoid to a large extent in-depth analysis of mass spectra.
Figure 1
Chemical treatment (Optional) Transfer of protein extracts Bacterial samples Direct transfer of bacterial colonies Sample preparation on MALDI target metal plate
Intensity
MALDI-TOF-MS
m/z MALDI mass spectrometry
Protein mass pattern detection Current Opinion in Microbiology
MALDI-TOF MS protein mass pattern detection method. Bacteria, grown under controlled conditions or enriched from biological material, are pretreated by simple chemicals or directly applied with MALDI matrix onto a metal target plate. The plate is introduced into a MALDI mass spectrometer. These steps and data acquisition and analysis can be automated. Highly abundant proteins or protein fragments are monitored simultaneously by mass spectrometry. The signals detected are generally in the range of about 1000–20 000 m/z (mass to charge ratio). As is shown, a typical mass spectrum provides a specific molecular-phenotypic bacterial trait, profile or fingerprint for identification. Usually, only positively charged peptides or proteins are detected. In general most of the analytes detected in this mass range by MALDI have a charge z of 1 (singly charged), or to a lesser degree a charge z of 2 (doubly charged). The figure items were taken from ScienceSlides SuiteTM 2007. Current Opinion in Microbiology 2012, 15:397–402
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information nor identification of proteins. Precise mass signals of a protein fragment or protein sequence data can, however, improve accuracy in identifying bacteria and in particular be used to gain additional information for detecting bacterial antibiotic resistance or virulence factors. These sophisticated analyses though widely used in discovery proteomics based on sequenced organisms [9], they are still inexpedient in practice of a routine diagnostic laboratory. Many of the mass signals detected from bacterial protein extracts are derived from conserved ribosomal or other abundant structural or regulatory house-keeping proteins [10]. Owing to the detection of integral and ubiquitous cellular proteins of fragments thereof, the method is sufficiently stable against environmental factors only if conditions are not changed significantly during sample preparation. Protein mass pattern spectra can be analysed for identification of bacteria on the genus, species, and potentially even on the subspecies level [11,12]. Although the current emphasis lies on bacterial detection, the approach described can potentially be extended for detection of other microorganisms such as mycobacteria [13], fungi [14], and yeast [15], while viral detection will remain difficult as viruses have for example relatively lower protein content in general.
Sample preparation Whole bacterial cells can be easily processed with minor amounts of work and involving low-cost consumables [18]. A number of methods have been developed to generate samples from a large range of different bacterial genera. Usually a single bacterial colony is sufficient for MALDITOF analysis. In many cases culturing of bacteria is required to obtain enough material. A broad range of (selective or non-selective) solid or liquid media known from conventional procedures including blood cultures can be applied to various microorganisms but may have to be optimised from case to case. Moreover, direct microbial identification from blood, urine other body fluid or stool samples can be performed without culturing using enrichment methods [19]. In practice, culturing can be advantageous to gain initial information on the samples by distinguishing phenotypic features such as morphology (cocci or bacilli), Gram staining, culture characteristics, or antibiotic susceptibility profiles. In general subculturing requires at least an additional day but can produce more reliable mass spectral data, as a result of increased amounts of bacterial samples and/or reduced complexity of polymicrobial cultures.
Bacterial identification by mass spectrometry is a simple process. Mass spectra of reference bacterial strains are stored as reference libraries. For identification, suitable algorithms are applied to compare mass spectral fingerprints derived from bacterial probes with reference spectra [16]. For example, by pattern-matching algorithms, peaks in experimental mass spectra (Figure 1) that match with reference spectra and vice versa are counted, and intensities of matched signals are correlated with each other. Similarity or other score values obtained from this analysis can be compared with experimental threshold values applied for reliable identification on the genus and the species level. For identification of closely related subspecies, more indepth analysis of mass signals can be applied in which additional values are assigned to particularly informative signals observed in the reference spectra of bacterial samples [16].
Differential mass spectra of the same bacteria can occur owing to dramatically varying culturing conditions or preparation methods. Initially, this has impeded the application of the method. But application of validated and well-controlled growth media and standardised sample preparation procedures enabled generation of reproducible mass spectra for routine bacterial identification [20,21]. In some refined protocols chemical pretreatment during protein extraction is used to prepare sufficient proteins and peptides for MALDI analysis. For inactivating highly pathogenic bacteria and spores, fast pre-treatment steps that use for example ethanol or trifluoro acetic acid (TFA) have been developed [22]. The most convenient ‘direct transfer’ method omits any pretreatment. In this method, a bacterial colony is smeared onto a MALDI target as a thin film and MALDI matrix solution is laid over the already dried bacterial samples. This way, using acidic organic MALDI matrix, proteins and peptides are extracted from bacteria via sample preparation on the target plate and made that accessible to efficient laser desorption/ionisation.
Furthermore, bacterial mass patterns can be used as phenotypic traits for classification using unsupervised hierarchical clustering or other approaches applied in taxonomy [17]. As described, MALDI mass spectrometry analysis of bacterial cells particularly detects abundant housekeeping proteins, especially ribosomal proteins. Thus, alternatively to sequence analysis of 16 S rDNA mass pattern data of these proteins can be used as taxonomic markers to generate dendrograms for visualisation: The more similar the mass spectra, the closer the relationships of the bacteria [1,18].
Standardisation of laboratory conditions is particularly important to maintain identification capability of subspecies with slightly different but informative mass patterns. A number of protocols have been optimised with regard to handling of (specific) microbial samples and fine-tuning of MALDI analysis parameters [18,23], as well as optimisation of protocols for handling of blood cultures to remove interfering blood cells and host proteins [24]. Furthermore, mass spectrometry based bacterial identification has been largely successfully validated for comparison with conventional automated bacterial identification techniques and
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could produce very convincing intralaboratory and interlaboratory reproducibility [25,26]. The ability to differentiate closely related bacteria by mass patterns might also be improved by increasing the number of peptides using in addition to protein extraction proteases such as trypsin to digest bacterial proteomes [27]. The resulting protein fragment patterns can be compared against reference mass spectra produced by protease digestion of bacterial reference protein extracts. Standardisation of this approach may be more challenging than simple protein extraction methods as the reproducibility of protease digestion of whole proteomes of bacterial cells can be undesirable in practice.
Software tools Mass spectra can be easily digitalised, which enables straightforward data storage and exchange. For microbial identification and taxonomic classification user-friendly commercially software packages such as the MALDI Biotyper provided by Bruker Daltonics (http://www.bdal.com) and Samaris developed by AnagnosTec (http://www. anagnostec.eu/) are currently mainly used in the community. Besides these two widely proven software packages, recent data analysis tools such as Andromas [28] provide algorithms for reliable identification and classification of protein mass patterns, as well as steadily up-to-date reference mass spectra of several thousands of bacterial and other microbial entries. Using these data analysis tools, researchers can also create and upload their own library of reference mass spectra [16,29].
Performance in practice MALDI-TOF mass spectrometry based bacterial identification has been validated during the past years in a broad range of bacteria and microorganisms. Depending on the bacterial genera or species, over 90% to close to 100% experimental reproducibility and identification success rates were reported [30]. The accuracy was achieved with turnover rates of less than an hour instead of about one or two days required for the conventional methods, and a significant shortening of technician working time for sample and data processing. Moreover, the cost for consumables is estimated to be reduced to a twentieth compared to biochemical methods, while the mass spectrometer used has slightly higher costs than automated systems for large-scale bacterial identification by API [31]. In general, API and MALDI-TOF MS of protein mass patterns produce similar results. In the case of human pathogens such as Bacteroides fragilis, mass spectrometry even outperformed conventional assays [32]. Validation experiments using in parallel the MALDI Biotyper (Bruker Daltonics) and the Samaris (AnagnosTec) software revealed slightly different results, mainly as a consequence of varying bacterial entries in the commercially available reference spectra databases [33– 35]. Adding reference spectra of microorganisms that are either under- or not at all represented and increasing the Current Opinion in Microbiology 2012, 15:397–402
number of available mass spectra per species will steadily improve the performance of the method [36]. Well-organised community efforts including large reference centres to produce publicly available databases of standardised bacterial reference spectra, may make the methodology more independent from the commercially available spectra libraries. More comprehensive highquality reference spectra may even increase identification accuracy and accelerate acceptance of the promising method. Extensive double-blind studies on a number of different microorganisms involving several laboratories will further validate mass spectrometry based microbial identification systems [37]. Consequently, current efforts of the aforementioned companies in the field focus in particular on certification of the method for diagnostic applications.
Mass spectrometry detection of microbial nucleic acids In contrast to the phenotypic detection of protein profiles described so far, genotypic methodologies such as DNA sequencing, tailored PCR-based or restriction-enzyme based methods can more easily provide sufficient resolution to identify bacterial strains, and can be used to detect efficiently viruses. Mass spectrometry can not only detect peptides and proteins but also nucleic acids. For example, bacteria can be identified to the species level by protein mass pattern detection, while using the same instrument more in-depth information, if necessary, can be gained by additional mass analysis of their nucleic acids. PCR amplification and MALDI-detection based SNP-genotyping [38–40], MALDI-based re-sequencing [41,42] or electrospray ionisation mass spectrometry detection of PCR products [43,44] are new alternatives for the analysis of nucleic-acids based microbial markers, which may allow efficient monitoring of microbial epidemics [45]. In contrast to protein profiles, mass spectrometry based sequence analysis of microbial genomes can in principle rapidly and accurately reveal known or emerging antibiotic resistance mediated by gene mutations, mobile genetic elements or virulence factors, and detect viruses. Owing to the specific amplification of targeted regions of nucleic acids by PCR, low levels of bacteria can be detected quantitatively, even in higher complexity samples. In general, purification of DNA from bacteria is required for efficient amplification as the reliability of amplification of nucleic acids from crude mixtures is low. PCR also implies that potentially falsepositive PCR products can interfere with the analysis because even single copies of bacterial DNAs can potentially lead to striking signals and misinterpretation.
Routine diagnostics Mass spectrometry based protein profiling can be readily applied as its workflow fits well with classical culturing procedures. Interestingly, short and simple morphological www.sciencedirect.com
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and phenotypic testing such as Gram staining or detection of bacterial cytochrome c oxidases and/or catalase activity can be combined with mass spectrometry to prevent major misidentifications. The sample preparation steps after culturing are easily applicable and can be performed in a few minutes using simple reagents or kits [46,47]. Commercial solutions for automated handling and tracking of bacterial samples are available tailored for mass spectrometry analysis of bacteria. As protein profiling is not challenging in terms of mass accuracy and signal resolution, simple high-throughput MALDI mass spectrometers with minor requirements on maintenance can be applied. Therefore, owing to the availability of easily applicable software tools, the degree of training required for a technician is relatively low.
3.
4. Anhalt JP, Fenselau: Identification of bacteria using mass spectrometry. Anal Chem 1975, 47:219-225. Development of the basic idea of using mass spectrometry for microbial diagnosctics. 5.
With the further development and certification of the protein mass pattern detection method, current and near future applications include mainly medical and veterinary diagnostics applications, monitoring of phytopathogens, and ensuring food safety and quality control as are required in pharmacology, cosmetics, drinking water purification, and industrial microbiology. In the immediate future, mass spectrometry will certainly improve routine microbial diagnostics.
Acknowledgements We would like to thank Drs. Markus Kostrzewa, Thomas Maier, and Jo¨rg Sauer for fruitful discussions, Anja Freiwald for support, and Dr. ChungTing Han for critically reading of the manuscript. Our work is funded by the German Ministry for Education and Research (BMBF, grant number 0315082), the European Union (FP7/2007-2013, under grant agreement n8 [HEALTH-F4-2008-201418] entitled READNA, and [FP7/2007-2011], under grant agreement n8 262055 (ESGI). We apologise to all those, whose work cannot be covered here owing to space limitations.
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