Microbe ID Intact Cell Mass Spectrometry

Rapid microbial analysis and identification by intact cell mass spectrometry using MALDI-TOF Mass spectrometry

Microbe analysis and identification by Intact Cell Mass Spectrometry :Microbe analysis and identification by Intact Cell Mass Spectrometry Martin Winkler, Ph.D. Consultant Biotech-advisor.com

Intact Microbial Identification and Analysis by MALDI-TOF Mass Spectrometry :Intact Microbial Identification and Analysis by MALDI-TOF Mass Spectrometry MALDI-TOF Mass spectrometry (MS) Need for Rapid Microbiology Analytical variables with Intact Cell (IC) MS Examples of IC-MS with two pathogenic bacteria Helicobacter pylori Campylobacters Identification of biomarker ions in bacterial species (compared with pattern recognition methods). Mechanism of the IC-MS phenomenon Commercial MS systems and databases for IC-MS

Need for Rapid Microbiology :Need for Rapid Microbiology Bioterrorism and Bio-warfare: Anthrax, smallpox, etc. Acute, deadly infections: Bact. Meningitis, Strep, Staph. Food safety, e.g. E. coli H7:O157 Environmental Hazards, e.g. Legionnaire’s disease Process monitoring in fermentors, bioreactors Rapid analysis of bacterial clones carrying recombinant constructs (molecular biology applications) Research applications where quick answers to questions about bacteria needed with generic technology.

MALDI-TOF MASS SPECTROMETRY :MALDI-TOF MASS SPECTROMETRY Developed by Karas and Hillenkamp in 1980s. Sample in acidic matrix absorbs laser light Laser light shown on (matrix + sample) ionizes sample Ions swept into electric field then flight tube. Charged ions detected at end of tube. Mass/charge = constant * time of flight squared Figure of merit for instrument is Resolution Resolution expressed in ppm or percent Resolution typically < 0.1% for macromolecules such as proteins. Protein, peptides, DNA, polymers analyzed. ^ Res.DE, Q-TOF, TOF-TOF analyzers

Slide 5:Applied Biosystems DE Voyager MALDI-TOF MS (from manufacturer’s product instrument manual).

Protein Spectra: MALDI TOF Mass spectrometry of Hepatitis Surface Antigen(Winkler, M.A., Xu, N., Wu, H., and Aboleneen, H. Anal. Chem. 4: 664A-667A (1999). :Protein Spectra: MALDI TOF Mass spectrometry of Hepatitis Surface Antigen(Winkler, M.A., Xu, N., Wu, H., and Aboleneen, H. Anal. Chem. 4: 664A-667A (1999).

MALDI-TOF MS: Analytical Parameters :MALDI-TOF MS: Analytical Parameters Spot 1-15 picomoles of analyte (nmol conc in 0.5-1 uL) Analyte depleted of NaCl and salts Analyte free of SDS or other ionic detergent. Analyte soluble in acid (e.g. 0.1% TFA) Matrix choice: e.g. Sinapinic acid in AcN/acid/water. Others are ferulic, ACCA; depends on positive or negative mode of analysis & analytes (pep.,prot., DNA, polymer, etc.) Matrix and sample mixed and dried Laser power set to lowest energy for ionization of analyte. Number of “shots” accumulated typically 100-150 with data smoothing.

Analytical Figures of Merit: MALDI-TOF MS :Analytical Figures of Merit: MALDI-TOF MS Resolution = ΔM/M (ΔM Full width half height). Expressed as % or ppm. Daily calibration and/or calibration check is necessary. Resolution typically < 0.1% Internal or external calibration used Signal to Noise ratio (S/N). Varies with number of shots accumulated, power of laser used, mass analyzed, presence of salts and other contaminants. Lower mass analytes easier to ionize (require less power to ionize).

Intact Cell Mass Spectrometry: Discovery :Intact Cell Mass Spectrometry: Discovery Bacterial mass spectrometry first described by Anhalt and Fenselau (1975). Lower mass (non-protein) ions described. IC-MS of bacterial species independently described by Jack Lay (NCTR-FDA) and Martin Claydon ( U. of Manchester). First discovered in analysis of lysed bacteria by MALDI-TOF MS (David Lubman group) Intact Cell controls ran in Lubman’s study of lysed bacteria: observed MALDI spectra with intact bacterial control and there was no detectable lysis of control bacteria. Spectrum of ions intact bacteria is a subset of those observed with lysed bacteria but spectra appeared to be unique and reproducible for each species described.

Intact Cell Mass Spectrometry Technique :Intact Cell Mass Spectrometry Technique Generic technique: Applicable to any bacterial species Intact Bacteria (~1-5 x 106 /ml) suspended in acidic and/or organic solvent Bacteria, then added to matrix (e.g., sinapinic acid in water/acetonitrile/0.1% TFA) Alternatively, bacteria or culture smeared on sample plate. Then matrix solution added. Lectin or affinity surfaces also used to capture bacteria from mix. ACCA matrix for ions of 1-15 kDal; sinapinic or ferulic for 10 kDal and higher mass range analytes. Analysis typically by MALDI-TOF MS in positive ion mode. Ions: Species-specific and non-specific Positive ions detected with M/z from 600 to >62,000 Most ions observed in range m/z 1-15,000

Analytical Variables: IC-MS :Analytical Variables: IC-MS Number of bacteria spotted (> 5 x 103) Source of bacteria (culture, colony, environment) Purity of bacteria (clone vs. mixed species). Removal of growth/culture media and salts Bacterial growth stage at harvest Age of cultures (and sterilization method) Organic/acid composition of sample diluent(s) Time before analysis Matrix type selected (ACCA, sinapinic, ferulic, etc.) Matrix solution comp. (MeOH, IPA, AcN, and type of acid) MALDI-TOF MS variables also apply (laser power, shot #, etc.)

Analytical Variables: IC-MS (continued) :Analytical Variables: IC-MS (continued) Optimal conditions as determined from studies in Steve Musser’s lab (FDA), Karin Wahl and others: Sterilization with 50% ethanol or 50% methanol Diluent acid TFA at 0.1%-2.5% final (formic acid or others also used) Acetonitrile at conc.>50% ACCA for ions 1-12 kDal Ferulic or sinapinic acid for ions >12 kDal Sandwich method of spotting provides high reproducibility

Analytical Characterizations of IC-MS Methods :Analytical Characterizations of IC-MS Methods Karen Wahl and her group at Northwestern Research Laboratories have characterized method for 6 species. Their method uses internal std. and was accurate (<0.1%) Method was reproducible between 3 days, 3 instruments, 3 preps. with the 6 species. Blind study was accurate for automated algorithm used for identification with biomarkers. Defined biomarkers for each microorganism and the number of biomarkers required to identify each species. Ed Reilly and co-workers also described spectrum pattern-matching methods for bacterial Identification with E. coli strains. Other groups have reported differentiating methicillin resistant E. coli and Staphylococcus strains by IC-MS and pattern recognition.

Intact Cell Mass Spectrometry: Examples with two Pathogenic bacteria from cultured colonies (Winkler, M., Uher, J. and Cepa, S. Anal. Chem. 71: 3416-3419 (1999) :Intact Cell Mass Spectrometry: Examples with two Pathogenic bacteria from cultured colonies (Winkler, M., Uher, J. and Cepa, S. Anal. Chem. 71: 3416-3419 (1999) Helicobacter pylori: Rapid method and species identified. Strains not identified. Campylobacters: Species Identification– c. coli, c. fetus, c. jejuni. Strains not examined. Biomarkers for H. pylori and Campylobacters defined. Use of 50% methanol to preserve clones before analysis Definition of matrix solution composition to obtain higher mass biomarkers.

Helicobacter pylori :Helicobacter pylori Most prevalent human bacterial pathogen >50% of population infected Peptic and duodenal ulcer agent Associated with Stomach cancer risk Strains associated with above risks Susceptible to anti-microbial therapy A number of diagnostic tests (also immunoassays for antibody to H. pylori) Can be detected by biopsy and culture of biopsy

Rapid IC-MS Study: Campylobacters and Helicobacters (Winkler, M., Uher, J. and Cepa, S. Anal. Chem. 71: 3416-3419 (1999) :Rapid IC-MS Study: Campylobacters and Helicobacters (Winkler, M., Uher, J. and Cepa, S. Anal. Chem. 71: 3416-3419 (1999) Cultured 2 species of Helicobacter (12 strains ) on blood agar medium Harvest at 3 days (> 1 mm colony) Suspended in 0.04 mL 50% methanol Dilution into 50% Acetonitrile, 0.1% TFA Within 1 h analyzed with sinapinic acid solution Solution contained AcN/MeOH/water 1:1:1 containing saturated sinapinic acid

MALDI-TOF MS SPECTRUM OF BLOOD AGARWinkler et al Anal. Chem. 71: 3416-3419 (1999) :MALDI-TOF MS SPECTRUM OF BLOOD AGARWinkler et al Anal. Chem. 71: 3416-3419 (1999)

IC-MS of Helicobacter pylori from picked colony :IC-MS of Helicobacter pylori from picked colony

IC-MS of Helicobacters: Results :IC-MS of Helicobacters: Results Three biomarkers near 13,293, 24,236, 58,268 m/z distinguished H. pylori from H. mustelae (which biomarker at 49,608 m/z ) The 12 strains of H. pylori could not be reproducibly distinguished with any ions/biomarkers (Nillson has destinguished from studies of lysed bacteria by MALDI). Putative Urease high molecular weight enzyme identified (61.2 kDal) in H. pylori

Campylobacters :Campylobacters C. jejuni Leading cause of food borne illness (poultry, seafood) C. coli and C. fetus also widespread Strain typing by culture or PCR methods No rapid method for species discrimination IC-MS tested Campylobacters species typing and distinction from H. pylori.

IC-MS of Campylobacter jejuni from picked colony :IC-MS of Campylobacter jejuni from picked colony

IC-MS of Campylobacter fetus from picked colony :IC-MS of Campylobacter fetus from picked colony

IC-MS of Campylobacter fetus--Zoom in of spectrum to show biomarker :IC-MS of Campylobacter fetus--Zoom in of spectrum to show biomarker

IC-MS of Campylobacter coli from picked colony :IC-MS of Campylobacter coli from picked colony

Biomarkers identifying H. pylori and Campylobacters :Biomarkers identifying H. pylori and Campylobacters

Conclusions: Campy and Helico :Conclusions: Campy and Helico Helicobacter and Campylobacter species identified and distinguished (at least 2 Biomarkers each) Strains of Helicobacter pylori not distinguished High mass ions (>30 kDa) detected Putative Urease beta-chain product identified in H. pylori (61.2 kDa) C. Fenselau and co-workers confirmed and extended work to show that fragment ions of IC-MS of H. pylori match those predicted from GenBank gene/protein database, e.g. for Urease 61.2 kDal.

Mechanism of IC-MS? :Mechanism of IC-MS? Bacteria not lysed by laser but the strong organics and acids can lyse bacteria. Spectra not typical of lysed bacteria however. After centrifugation, supernatant fluids of bacterial AcN/water extracts also produce ion spectra of bacteria. Markers are primarily proteins extracted from cells or from cell walls. A number now identified structurally (J. Lay Lab, FDA). Abundant proteins preferentially detected (e.g. ribosomes, cell wall glycopeptides) Highly positively charged proteins (arg containing) most abundant in spectra Recombinant proteins: soluble vs. insoluble. Only water soluble proteins detected.

Commercial Systems for IC-MS :Commercial Systems for IC-MS Bruker Daltonics sells Biotyper MALDI MS system (www.bdal.com). Click on Biotyper Waters sells Micro MALDI TOF MS system (www.waters.com) click on mass spectrometry Other commercial systems (e.g. Applied Biosystems, Bio-Rad) amenable to analysis with consultation of literature methods & published spectra. High through-put and robotic systems available from above vendors (and others such as Bio-Rad) for colony picking, matrix spotting, sample handling.

IC-MS Microbes Analyzed to date (2007) :IC-MS Microbes Analyzed to date (2007) Hundreds of bacterial species’ spectra published Some strains distinguished (e.g. E. coli strains (Arnold and Reilly, 1998); Methicillin resistant strains) Anthrax and other bacterial spores differentiated. Intact Viruses analyzed (Fenselau group). Intact Yeast analyzed (Fenselau group). Fungal spores analyzed (Aspergillus) Field portable device developed by Johns Hopkins Advance Projects Lab (APL) for bioweapons detection (e.g. Anthrax detection) IC-MS still not a clinically validated method (RUO)

On-going work and challenges :On-going work and challenges Mixtures of bacteria analyzed (K. Wahl group) C. Fenselau group used affinity surfaces to specifically trap bacteria. Also proteolytic digestion of bacteria produced ions matched to bacterial gene and protein databases. Others have used lysozyme for Gram pos. Ion identification through structural analysis of ions (CID) in Q-TOF MS or TOF-TOF MS.

Resources for methods and data analysis :Resources for methods and data analysis Analytical services available for sample submissions if instrument is not available (e.g. Proteogene, CBI, Mass Consortium) Bruker, Waters, Applied Biosystems have on-line application notes at their websites associated with mass spectrometry systems. On-line database of IC-MS for research use (www.baktron.net) with ions and bibliography.

References: First Half :References: First Half Anhalt, J.P. and Fenselau, C. Identification of bacteria using mass spectrometry. Anal. Chem. 47:219-225. Arnold, R.J. and Reilly, J.P. Fingerprint matching of E. coli strains … using a modified correlation approach. Rap. Comm. Mass. Spect. 12: 630-636 (1998). Holland, R.D., Wilkes, J.G., Rafii, F., Sutherland, J.B., Persons, C.C.Voorhees, K.J. and Lay, J.O. Rapid identification of intact whole bacteria based on spectral patterns using MALDI-TOF MS. Rap. Comm. Mass. Spect. 10:1227-1232 (1996). Winkler, M.A., Uher, J., and Cepa, S. Direct Analysis and Identification of Helicobacter and Campylobacter species by MALDI-TOF Mass Spectrometry. Analytical Chemistry 71:3416-3419 (1999). Winkler, M.A., Xu, N., Wu, H., and Aboleneen, H. MALDI-TOF MS of Chemically modified Recombinant Hepatitis B Surface Antigens. Analytical Chemistry 4: 664A-667A (1999).

Summary: IC-MS for Microbe Identification :Summary: IC-MS for Microbe Identification Bacteria and spores typed; some labs report strain differentiation (E. coli strains, S. aureous Methicillin resistance.) Method is rapid (<0.5 hour), reproducible, and rugged between days and instruments. Method is sensitive, requiring several thousand organisms usually from cultured colonies or homogeneous cultures. Ions desorbed by MALDI tend to be smaller, positively charged proteins or glycoprotein or protein conjugates; many shared between species (e.g. ribosomal proteins) Biomarkers (two or more) are required. Pattern recognition and cross-correlation has also been used Dedicated commercial systems available (but not required). Protein/gene databases can be applied to identify ions. Second half: IC-MS for recombinant protein analysis