MALDI - TOF MS: Where Mass Spectrometry Meets Microbiology

Abstract:
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; MALDI biotyping) has turned a promising tool for the accurate, rapid, and economical identification of pathogens in the clinical diagnostics laboratory. MALDI-TOF measures the mass of molecules from a sample that has been embedded in a matrix by using a laser to ablate and desorb the molecules with minimal fragmentation. The mass spectrum is produced from the pattern (i.e., position and relative intensity) of detected m/z peaks, generating a distinct profile for a particular sample. The method is continuously being improved, and new applications for distinguishing strains, identifying metabolites or functional characteristics (e.g., antibiotic resistance), and detecting microbes directly in host samples have been developed. Adopting these methods in other disciplines than clinical diagnostics, for example, in agriculture, food safety and quality testing, or ecology, will open up new opportunities for diagnostics and research. Nowadays, MALDI-TOF MS technology is most widely used for discovering potential cancer biomarkers. MALDI-TOF MS presents advantages as it is easy to use the technique with high automation and throughput potential and high sensitivity. New fields of application for MALDI-TOF MS, such as Surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF MS) and imaging mass spectrometry (IMS) are promising tools in the field of oncology.

1. MALDI - TOF MS Where Mass Spectrometry Meets Microbiology Credit Seminar - I Chair Dr. K.V. RAJENDRAN Principal Scientist, HOD, AEHMD ICAR- CIFE (Deemed University) Presented by B. MADHUSUDHANA RAO AAHM-PA7-01

2. INTRODUCTION: Microbiology The identification of microorganisms - h as long been mainly based on the detection of phenotypic characteristics exhibited by the putative pathogens. Gram stain, colony morphology, microscopic examination, Vitek 2, Microscan , numerous API methods, disks on media, growth characteristics, selective media, chromogenic media, biochemical tests, serologic tests, enzymatic reactions, with either manual or automated methods , form the mainstay of the classification of bacteria, yeast and fungi. Complete identification requires at LEAST TWO DAYS , or more for fastidious organisms.

3. MICROBIAL IDENTIFICATION 1990 1970 2000

4. What are the Recent advances in Identification of pathogens Rapid nucleic acid amplification methods such as real-time PCR using melting curve analysis, multiplex PCR, fluorescence in situ hybridization (FISH ) and peptide nucleic acid- FISH (PNA-FISH). These assays, however, only target specific organisms; require technical expertise ; and specimens are usually processed in batches. MALDI-TOF MS, a CHEMOTAXONOMIC method , also allows rapid identification of bacteria. Recently, the platform has successfully been used to improve veterinary diagnostics and to classify environmental isolates.

5. Features of MALDI-TOF MS Soft ionization - analyze intact biomolecules and synthetic polymers Broad mass range - analyze a wide variety of biomolecules Simple mixtures are okay Relatively tolerant of buffers and salts Fast data acquisition Easy to use and maintain , no water or gas hook ups required High sensitivity, superior mass resolution and accuracy (MALDI-TOF MS) is a EMERGING TECHNOLOGY in Diagnostic Microbiology Rapid and cost effective identification of bacteria directly from isolated colonies and positive culture bottles. Protein biomarkers measured are highly expressed proteins responsible for housekeeping functions, such as ribosomal (16S) and transcription/translation factor proteins

6. Comparative studies on identification of microorganisms using different MALDI-TOF MS platforms and databases from the time point of the published work.

7. ADVANTAGES: MALDI and TOF Gentle Ionization technique High molecular weight analyte can be ionized Molecule need not be volatile Sub-picomole sensitivity easy to obtain Wide array of matrices Best type of mass analyser to couple to MALDI Unlimited mass range Macromolecules > 400,000 – accurately measured 100 pulses of laser light (10nanoseconds) Result – seen as a good spectrum Camera – tracks the laser beam around the MALDI M atrix- A ssisted L aser D esorption I onization - T ime o f F light M ass S pectrometry

8. Principles of MALDI-TOF MALDI is s o f t ionization technique used in ma s s spectrometry, allowing the analysis of biomolecules such as DNA, proteins, peptides & sugar or polymers such as dendrimers and macromolecules It is three steps method. The sample is mixed with a suitable matrix & applied to a metal plate. A pulsed laser irradiate a sample triggering desorption of matrix material . Ionization of analyte molecules Animation: http: /cmgm.stanford.edu/pan/section_html/MS/

9. Principles of MALDI-TOF Mass Spectrmetry Sample molecule Matrix molecule Sample embedded in light-absorbing matrix Excitation of matrix molecules by laser light Laser De s o r ptio n / p r o t o n a ti o n of sample molecules MH +

10. MALDI-TOF MS: History Developed in 1980s by Karas and Hillenkamp Term was coined in 1985 by Franz Hillenkamp , Michael Karas Detection of large molecules using TOF by Tanaka and Yoshida First commercial database developed by Anagnostec (1998 ) Shimadzu scientist receives Nobel Prize in Chemistry – Kiochi Tanaka (2002) Technology in use in Europe for >10 years.

11. MALDI-TOF MS Overview des o rpti o n i o n i za t i o n accelerati o n separation de t ecti o n m z = 2eU L² t² m: mass z: charge U: acceleration voltage L: path length t: time e: elementary charge + + + + t a rge t cr y st a ls acceleration zone ring electrode ion detector Uncharged Drift region Time of Flight V a c u u m tube + + matrix/analyte Which protein molecules? Those that are easily desorbed, Like ribosomal proteins Multiple LASER shots Absorbs e from LASER Time of flight is a function of the specific ion mass (m/z) Soft ionisation method: low level of sample fragmentation

12. MALDI TOF Sample Preparation Spot target slide with direct colony (can be up to 5 days old). Bacteria, molds , yeasts, mycobacteria Air dry for 1-2 min. Target Slide 48 wells STEP 1 STEP 2 STEP 3 STEP 3 Load target slides Create Spectra Matrix Solutio n (5µl α -cy ano-4-hydroxycinnamic acid)

13. MALDI-TOF MS : Sample preparation Direct transfer spreading of intact cells directly onto a steel plate lysis of cells occurs during the contact with acid matrix and by laser desorption most bacteria Proteins extraction previous extraction of proteins by organic acids and/or alcohol ( e.g. ethanol and 70% formic acid ) yeasts, moulds, some species of bacteria (depending on the cell wall composition) 2) MALDI-TOF MS analysis unique mass spectral fingerprint of desorbed microbial cell components ( mainly intracellular proteins ), different among genera , species or also some strains http://cmr.asm.org/content/26/3/547.figures-only 3) identification : comparison of mass spectrum to those of reference strains in database

14. Sample preparation Direct Smear Pick colony Smear onto target Allow to dry Add matrix Allow to dry Analyse Extraction Pick colony Resuspend Add ethanol Inactivation/storage/shipment Add formic acid and ACN Centrifuge Pipette supernatent onto target Allow to dry Add matrix Allow to dry Analyse

15. Direct Detection for Positive Blood Culture by MALDI TOF Journal of Clinical Microbiology 51;805-809, 2013 Bruker S e psityper Kit Journal of Clinical Microbiology 48;1584-1591, 2010 Issues: Removal of host proteins Extraction protocol required Bacterial concentration need~10 7 /mL Polymicrobial specimens Seen on Gram stain? Antibiotic resistance genes Yeasts?

16. MALDI-TOF MS: MATRIX PREPARATION http://cmr.asm.org/content/26/3/547.figures-only Matrix: able to absorb the energy of the laser (usually 337 nm) able to crystalise with samples within seconds – necessary for sample desorption usually acid character (proton ionisation of sample), dissolved in organic solvent H CHC: α -Cyano-4-hydroxycinnamic acid (organic solvent: 50% acetonitrile with 2,5 % trifluoracetic acid) . The OS is used in making up the Matrix and the bacterial test standard (BTS). Add 250 µl "OS" to one tube of "HCCA matrix portioned" and vortex until all matrix crystals are completely dissolved, this may take several minutes but is important to completely dissolve. Prepared matrix MUST be stored in the dark at room temperature and can be viable for up to 2 weeks (“best before”). SA : 3,5-Dimethoxy-4-hydroxycinnamic acid (sinapic acid) ; DHB : 2,5-Dihydroxybenzoic acid http://www.sigmaaldrich.com/catalog/product/sigma/c8982?lang=en&region=CZ

17. Sample preparation based on type of organism:

18. Potential Options for Direct detection from clinical specimens Clark A E et al. Clin. Microbiol. Rev. 2013;26:547-603

19. MALDI-TOF MS: ANALYSIS 19 Comparison of mass spectrum protein profile of unknown sample with these of reference strains present in database by software Commercial databases from different MALDI-TOF MS producers Bruker Daltonics – MALDI BIOTYPER Shimadzu - Shimadzu Launchpad software + SARAMIS database Biomérieux - VITEK® MS Other databases compatible with different hardware systems ( e.g . Andromas ) BioTyper: The statistical analysis for correlation includes peak positions , intensities and frequencies across the complete range of microorganisms. Score value: 0 (none similarity) - 1000 (absolute similarity) But it is expressed in decadic logarithm log(score value): 0-3 Range Description Symbols Color 2.300 ... 3.000 highly probable species identification ( +++ ) green 2.000 ... 2.299 secure genus identification, probable species identification ( ++ ) green 1.700 ... 1.999 probable genus identification ( + ) yellow 0.000 ... 1.699 not reliable identification ( - ) red

20. Select a Colony Smear a Thin-Layer onto a MALDI Target Plate Unknown Mic r oo r ganism Add MALDI Matrix Identified Species MALDI Biotyper Workflow MALDI Biotyper Data Interpretation Generate MALDI-TOF Profile Spectrum

21. A Rapid method to Investigate Bacteremia and Septicemia Bruker‘s MALDI Sepsityper enables identification of gram- negative bacteria, gram-positive bacteria and yeast from positive blood cultures within 30 minutes. mainly ribosomal proteins : further cold-shock and heat-shock proteins, chaperons etc.

22. Biochemical Tests API Rapid ID Methods Automated Identification Real-Time PCR MALDI-TOF MS Sensitive and Specific ☺ ☺☺ ☺☺ ☺☺ ☺☺ ☺☺☺☺ Rapid ☺☺☺ ☺ ☺☺ ☺☺☺☺ Easy to perform ☺☺ ☺☺ ☺☺☺ ☺☺☺ ☺☺ ☺☺☺ Easy to interpret ☺ ☺☺ ☺☺ ☺ ☺☺☺☺ Cost Effective ☺☺☺ ☺☺ ☺☺☺ ☺☺ ☺ ☺☺☺☺ High Through-Put ☺☺ ☺☺☺ ☺☺ ☺☺☺ Use with multiple organism types ☺ ☺☺ ☺ ☺☺☺ Able to interface to LIS ☺☺ ☺ ☺☺ Can be automated ☺☺ ☺☺☺☺ MALDI-TOF Vs. Current Identification Methods

23. APPLICATIONS Rapid turn around time, high throughput - more accurate & cheaper than other procedures based on biochemical tests . - impact on appropriate emperic therapy Broad applicability (all types bacteria including anaerobes, yeasts, fungi) Pesticides on foods Soil and groundwater contamination and toxin quantification. Ability to resolve poly-microbial specimens AMR determination - (already MRSA, carbapenemase ) Strain typing Single colony requirement - direct from blood culture Low exposure risk –sample inactivation COST SAVINGS

24. Advantages & Trends of change with MALDI-TOF-MS MALDI-TOF-MS is a rapid, precise, and cost-effective method for identification of intact bacteria, compared to conventional phenotypic techniques or molecular biology. Furthermore, it allows identification of bacteria directly from clinical samples (blood cultures for example) Rapid identification ~ 1min per isolate Reduced cost per test Cost will be <$1.50 per determination Reduced Hands-on-Time Tech setup time 2-3 minutes Flexibility - each bench get their own target slide High throughput – 192 isolates/run

25. Limitations: Databases : still in their infancy High initial capital expenditure New approaches (business models) Potential instrument downtime - single instrument E. coli Vs. Shigella Very closely related and cannot be differentiated Molecular methods Streptococcus pneumoniae Vs. Streptococcus mitis group Very closely related, new databases can give a definitive ID Differentiate by Bile solubility or optichin disk No Test Is Perfect

26. CASE STUDIES: All the freshly caught fish obtained from fishermen exhibit satisfactory microbiological quality with crucian carp ( Carassius carassius ) was the most contaminated fish among all the tested species. The Pseudomonas spp. was predominant in fish microflora in all cases. Pseudomonas spp. cause rapid deterioration of fish and fish products. Genomic analysis only identified the species of 50% of the isolated strains , proving to be particularly poor at identifying members of the Pseudomonas and Bacillus genera. In contrast, MALDI-TOF MS fingerprinting identified 76% of the strains at the species level . The mass spectral data were submitted to the Spectra Bank database ( http://www.spectrabank.org ). Furthermore, cluster analysis of the peak mass lists was carried out with the web application SPECLUST and the calculated groupings were consistent with results determined by a phylogenetic approach that is based on the 16S rRNA sequences.

27. SELDI-TOF-MS

28. IMS – Looking beyond Classical Histology New fields of application for MALDI-TOF MS, such as imaging mass spectrometry (IMS) could be implemented in the near future. IMS allows the analysis of molecules directly from tissue sections and has been applied to address questions about skin pathophysiology, drug absorption, and metabolism. This tool allows direct visualization of the spatial distribution of biomolecules in biological sections in a single run without radioisotope or fluorescence labeling . IMS technology is well-positioned to study a wide variety of chemical interactions, including those which occur inside single-species microbial communities, between cohabitating microbes , and between microbes and their hosts. The capacity for the profiling of molecules directly from tissue samples by MALDI-TOF MS would open an attractive investigation field in different areas such as nanomedicine, pharmacokinetics, proteome imaging etc.

29. MALDI Imaging Workflow Tis s ue Cryosection Matrix coating MALDI Mass Spectrometry H&E staining

30. APP L IC A TIO N S MALDI – TOF MS Chemistry Taxonomy Microbiology C linic a l a n a lysis Pharmaceutical analysis APPLICATIONS

31. CONCLUSION: Rapid, precise, and cost-effective method for identification of intact bacteria, compared to conventional phenotypic techniques or molecular biology. Direct identification of pathogens in samples Search for antibiotic resistance determinants and virulence factors, and antibiotic susceptibility testing Manual colony picking is a major limitation of the throughput of MS identification and causes major errors due to inversions, which are estimated to occur at a rate of up to 3%. As MALDI-TOF MS is currently used to identify more than 95% of all isolates in laboratories. MALDI COLONYST is the latest generation intelligent robot optimized for colony picking and complete MALDI target preparation. Limited reference spectra in database for some genera and species Identifications will get better. Can be automated. NEXT BIG CHANGE IN CLINICAL MICROBIOLOGY.

32. Benagli C., Rossi V., Dolina M., Tonolla M., Petrini O., 2011. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the identification of clinically relevant bacteria. PLoS One 6:e16424 Samuels G.J., Ismaiel A., Bon M.C., De Respinis S., Petrini O., 2010. Trichoderma asperellum sensu lato consists of two cryptic species. Mycologia , 102:944–966 Mellmann A., Cloud J., Maier T., Keckevoet U., Ramminger I., Iwen P., Dunn J., Hall G., Wilson D., Lasala P., Kostrzewa M., Harmsen D., 2008. Evaluation of matrixassisted laser desorption ionization-time-of-flight mass spectrometry in comparison to 16S rRNA gene sequencing for species identification of nonfermenting bacteria. Journal of Clinical Microbiology, 46:1946–1954. Böhme , K., Fernández‐No, I.C., Pazos , M., Gallardo, J.M., Barros‐Velázquez, J., Cañas , B. and Calo ‐Mata, P., 2013. Identification and classification of seafood‐borne pathogenic and spoilage bacteria: 16 S r RNA sequencing versus MALDI‐TOF MS fingerprinting.  Electrophoresis ,  34 (6), pp.877-887. Bizzini , A. and Greub , G., 2010. Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry, a revolution in clinical microbial identification.  Clinical Microbiology and infection ,  16 (11), pp.1614-1619. References

33. References Identification of Bacteria in Blood Culture Broths Using Matrix- Assisted Laser Desorption-Ionization Sepsityper ™ and Time of Flight Mass SpectrometryJen Kok,1,2,* Lee C. Thomas,1 Thomas Olma,1 Sharon C. A. Chen,1 and Jonathan R. Iredell1, PLoS One. 2011; 6(8): e23285. NCBI resource Ashim k. Chakravarty - Intoduction to Biotechnology – 2013- Oxford university press – New Delhi – Pg. no : 301 Glazer N. Alexander, Hiroshi Nikaido – Fundamentals of Applied Microbiology – second edition – 2008 – Cambridge university press - Pg. no: 159 – 162 H,K. Das – Textbook of Biotechnology – fourth edition - 2011- Beekem printers – NewDelhi - Pg. no: 135. H.S. Chawla – Introduction to Plant Biotechnology – third edition – 2013 – Oxford IBH Publishing company pvt ltd – NewDelhi - Pg. no : 589 – 590 Keith Wilson, John Walker – Practical Biochemistry, Principles and Techniques – fifth edition – 2000 – Cambridge university Press - Pg. no : 592 – 595, 586, 602 P.K. Gupta – Biotechnology and Genomics – first edition – 2010 – Rastogi publications – Meerut - Pg. no : 83 - 87

34. References Rahi , P., Prakash, O. and Shouche , Y.S., 2016. Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) based microbial identifications: challenges and scopes for microbial ecologists.  Frontiers in microbiology ,  7 , p.1359. Pavlovic, M., Huber, I., Konrad, R. and Busch, U., 2013. Application of MALDI-TOF MS for the identification of food borne bacteria.  The open microbiology journal ,  7 , p.135. Cho YT, Su H, Wu WJ, Wu DC, Hou MF, Kuo CH, et al. Biomarker characterization by MALDI-TOF/MS. Adv Clin Chem. 2015;69:209–54. Gekenidis MT, Studer P, Wüthrich S, Brunisholz R, Drissner D. Beyond the matrix-assisted laser desorption ionization (MALDI) biotyping workflow: in search of microorganism-specific tryptic peptides enabling discrimination of subspecies. Appl Environ Microb . 2014;80(14):4234–41. Ng, W., 2013. Teaching microbial identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and bioinformatics tools.  Journal of Microbiology & Biology Education: JMBE ,  14 (1), p.103. Drissner , D. and Freimoser , F.M., 2017. MALDI-TOF mass spectroscopy of yeasts and filamentous fungi for research and diagnostics in the agricultural value chain.  Chemical and Biological Technologies in Agriculture ,  4 (1), p.13.

35. References Bizzini , A., Durussel , C., Bille , J., Greub , G. and 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.  Journal of clinical microbiology ,  48 (5), pp.1549-1554. Yang, J.Y., Phelan, V.V., Simkovsky , R., Watrous, J.D., Trial, R.M., Fleming, T.C., Wenter , R., Moore, B.S., Golden, S.S., Pogliano , K. and Dorrestein , P.C., 2012. Primer on agar-based microbial imaging mass spectrometry.  Journal of bacteriology ,  194 (22), pp.6023-6028. Clark, A.E., Kaleta , E.J., Arora, A. and Wolk , D.M., 2013. Matrix-assisted laser desorption ionization–time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology.  Clinical microbiology reviews ,  26 (3), pp.547-603. Buchberger , A.R., DeLaney, K., Johnson, J. and Li, L., 2017. Mass spectrometry imaging: a review of emerging advancements and future insights.  Analytical chemistry ,  90 (1), pp.240-265.

36. SUGGESTIONS PLEASE