1887

Teaching Microbial Identification with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and Bioinformatics Tools

    Author: Wenfa Ng1
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    Affiliations: 1: Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117576
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Published 06 May 2013
    • Supplemental materials available at http://jmbe.asm.org
    • Corresponding author. Mailing address: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576. E-mail: ngwenfa@alumni.nus.edu.sg.
    • ©2013 Author(s). Published by the American Society for Microbiology.
    Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 103-106. doi:10.1128/jmbe.v14i1.494
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    Abstract:

    Ever since the first observation of “animalcules” under a microscope, and the subsequent discovery of microorganisms of myriad size, shape, pigmentation and motility modes, classification in aid of microbial identification is key to understanding inter-relationships between diverse microbes. Combining universal applicability with robustness, 16S rRNA sequencing is the gold standard for microbial typing; however, recent developments in clinical diagnostics have called attention to a shift towards PCR-independent instrumentation and methods given PCR’s requirement for expensive and complex sample preparation. Using ribosomal proteins as biomarkers for evolutionary relatedness, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) - originally developed for the soft ionization of proteins and peptides in proteomics studies - has been successfully applied to identifying bacteria, archaea, fungi and viruses to the species, and, on occasions, sub-species level. Though experimentally proven and increasingly adopted in the clinic, the relatively low-cost (on a per sample basis) and rapid MALDI-TOF MS microbial identification technique, along with its theoretical principles and methodology, is a conspicuous absentee in contemporary microbiology curricula. Motivated by a desire to close the curriculum gap, this article describes a discovery-based activity for teaching microbial identification - using MALDI-TOF MS in combination with open-source genomics and proteomics search tools – while providing tips on mass spectra interpretation and activity implementation for lowering the barrier for classroom adoption. Infused with inquiry-based learning concepts guiding students in identifying microbes from environmental water samples with unknown species diversity, the activity spurs students’ learning by igniting their spirit of inquiry, which leads to better mastery of concepts; a significant departure from conventional laboratory exercises that, in verifying known theory or results, lack the ability to excite students or impart skills for seeking answers to their own questions. Suitable as part of a laboratory sequence complementing bioinformatics, analytical chemistry or life sciences courses, the activity’s modular nature also affords creative content adjustments for catering to differing curricula needs and learning styles. Nor is the exercise restricted to water samples, other samples (with appropriate modifications in inoculation protocols) such as vegetable leaves are also interesting microcosms for performing microbial censuses. Taken together, from sample collection to cultivation and microbial typing, students engage in an integrated exercise designed to impart conceptual knowledge and practical skills – while unlocking the joy of learning and discovery – by answering a research question with unknown answers: what are the microorganisms present in an environmental water sample?

Key Concept Ranking

Ionization Mass Spectrometry
0.63325137
16s rRNA Sequencing
0.59371024
0.63325137

References & Citations

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2. Bascom-Slack CA, Arnold AE, Strobel SA 2012 Student-directed discovery of the plant microbiome and its products Science 338 485 486 10.1126/science.1215227 23112324 http://dx.doi.org/10.1126/science.1215227
3. Bessède E, et al 2011 Matrix-assisted laser-desorption/ionization biotyper: experience in the routine of a university hospital Clin Microbiol Infect 17 533 538 10.1111/j.1469-0691.2010.03274.x http://dx.doi.org/10.1111/j.1469-0691.2010.03274.x
4. Bizzini A, Durussel C, Bille J, Greub G, Prod’hom G 2010 Performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory J Clin Microbiol 48 1549 1554 10.1128/JCM.01794-09 20220166 2863943 http://dx.doi.org/10.1128/JCM.01794-09
5. Cherkaoui A, et al 2010 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 48 1169 1175 10.1128/JCM.01881-09 20164271 2849558 http://dx.doi.org/10.1128/JCM.01881-09
6. Croxatto A, Prod’hom G, Greub G 2012 Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology FEMS Microbiol Rev 36 380 407 10.1111/j.1574-6976.2011.00298.x http://dx.doi.org/10.1111/j.1574-6976.2011.00298.x
7. DeAngelis KM, et al 2011 PCR amplification-independent methods for detection of microbial communities by the high-density microarray PhyloChip Appl Environ Microbiol 77 6313 6322 10.1128/AEM.05262-11 21764955 3187179 http://dx.doi.org/10.1128/AEM.05262-11
8. Demirev PA, Ho Y-P, Ryzhov V, Fenselau C 1999 Microorganism identification by mass spectrometry and protein database searches Anal Chem 71 2732 2738 10.1021/ac990165u 10424165 http://dx.doi.org/10.1021/ac990165u
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11. Korfiatis KJ, Tunnicliffe SD 2012 The living world in the curriculum: ecology, an essential part of biology learning J Biol Educ 46 125 127 10.1080/00219266.2012.715425 http://dx.doi.org/10.1080/00219266.2012.715425
12. Merkel S 2012 The development of curricular guidelines for introductory microbiology that focus on understanding J Microbiol Biol Educ 13 32 38
13. Nicolaou N, Xu Y, Goodacre R 2012 Detection and quantification of bacterial spoilage in milk and pork meat using MALDI-TOF-MS and multivariate analysis Anal Chem 84 5951 5958 10.1021/ac300582d 22698768 http://dx.doi.org/10.1021/ac300582d
14. Reasoner DJ, Geldreich EE 1985 A new medium for the enumeration and subculture of bacteria from potable water Appl Environ Microbiol 49 1 7 3883894
15. Schumaker S, Borror CM, Sandrin TR 2012 Automating data acquisition affects mass spectrum quality and reproducibility during bacterial profiling using an intact cell sample preparation method with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Rapid Commun Mass Spectrom 26 243 253 10.1002/rcm.5309 22223309 http://dx.doi.org/10.1002/rcm.5309
16. Šedo O, Voráč A, Zdráhal Z 2011 Optimization of mass spectral features in MALDI-TOF MS profiling of Acinetobacterspecies Syst. Appl. Microbiol 34 30 34 10.1016/j.syapm.2010.11.008 http://dx.doi.org/10.1016/j.syapm.2010.11.008
17. Seng P, et al 2009 Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry Clin Infect Dis 49 543 551 10.1086/600885 19583519 http://dx.doi.org/10.1086/600885
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21. Wynne C, Fenselau C, Demirev PA, Edwards N 2009 Top-down identification of protein biomarkers in bacteria with unsequenced genomes Anal Chem 81 9633 9642 10.1021/ac9016677 19883058 http://dx.doi.org/10.1021/ac9016677
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2013-05-06
2017-09-25

Abstract:

Ever since the first observation of “animalcules” under a microscope, and the subsequent discovery of microorganisms of myriad size, shape, pigmentation and motility modes, classification in aid of microbial identification is key to understanding inter-relationships between diverse microbes. Combining universal applicability with robustness, 16S rRNA sequencing is the gold standard for microbial typing; however, recent developments in clinical diagnostics have called attention to a shift towards PCR-independent instrumentation and methods given PCR’s requirement for expensive and complex sample preparation. Using ribosomal proteins as biomarkers for evolutionary relatedness, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) - originally developed for the soft ionization of proteins and peptides in proteomics studies - has been successfully applied to identifying bacteria, archaea, fungi and viruses to the species, and, on occasions, sub-species level. Though experimentally proven and increasingly adopted in the clinic, the relatively low-cost (on a per sample basis) and rapid MALDI-TOF MS microbial identification technique, along with its theoretical principles and methodology, is a conspicuous absentee in contemporary microbiology curricula. Motivated by a desire to close the curriculum gap, this article describes a discovery-based activity for teaching microbial identification - using MALDI-TOF MS in combination with open-source genomics and proteomics search tools – while providing tips on mass spectra interpretation and activity implementation for lowering the barrier for classroom adoption. Infused with inquiry-based learning concepts guiding students in identifying microbes from environmental water samples with unknown species diversity, the activity spurs students’ learning by igniting their spirit of inquiry, which leads to better mastery of concepts; a significant departure from conventional laboratory exercises that, in verifying known theory or results, lack the ability to excite students or impart skills for seeking answers to their own questions. Suitable as part of a laboratory sequence complementing bioinformatics, analytical chemistry or life sciences courses, the activity’s modular nature also affords creative content adjustments for catering to differing curricula needs and learning styles. Nor is the exercise restricted to water samples, other samples (with appropriate modifications in inoculation protocols) such as vegetable leaves are also interesting microcosms for performing microbial censuses. Taken together, from sample collection to cultivation and microbial typing, students engage in an integrated exercise designed to impart conceptual knowledge and practical skills – while unlocking the joy of learning and discovery – by answering a research question with unknown answers: what are the microorganisms present in an environmental water sample?

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Figures

Image of FIGURE 1

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FIGURE 1

Overview of the various components—and key learning objectives—of the laboratory exercise. Combining cell culture, mass spectrometry, and bioinformatics analysis of biomarker peaks from cultivated microorganisms, students engage in an inquiry-based exercise using the scientific method to tackle a research question with unknown answers.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 103-106. doi:10.1128/jmbe.v14i1.494
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Image of FIGURE 2

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FIGURE 2

Concept underlying MALDI-TOF MS-based identification of microorganisms, in particular, differences in ratios and relative intensities of the mass peaks afford the ability to distinguish between species. Note the need for a culture step prior to mass spectrometry analysis, a key difference from the culture-independent approach of PCR amplification followed by 16S rRNA sequencing.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 103-106. doi:10.1128/jmbe.v14i1.494
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Image of FIGURE 3

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FIGURE 3

Steps important to the identification of mass peaks—using the proteome database search approach—from MALDI-TOF mass spectrum of individual colony. Briefly, MBGD stores both the nucleotide and amino acid sequence of each ribosomal gene—where accessing the gene’s UniProt page yields the ribosomal protein’s molecular weight (and also ratio; assuming +1 molecular ion charge), which, in turn, can be used to search the extracted peaks list of each mass spectrum.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 103-106. doi:10.1128/jmbe.v14i1.494
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