Abstract
Short wet heat-treatment is presented as a new technique to release high-mass biomarkers to obtain strain-specific fingerprints of intact bacterial spores by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Wet heat-treatment was applied for several minutes (3–30) by two techniques using either a screw-cap tube submerged in a glycerol bath at 120 °C or an Eppendorff-tube submerged in a water bath at 100 °C. Both techniques turned out to be successful for releasing high-mass biomarkers. The influence of different experimental parameters and microbiological handling on the peak pattern of the released high-mass biomarkers was studied. While the sporulation medium, the applied washing procedure, and the choice of matrix crucially influenced the peak pattern, other parameters like storage conditions were found to be insignificant. A protocol of optimized experimental conditions for MALDI-MS of wet heat-treated spores is presented.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Anhalt, J. P.; Fenselau, C. Identification of Bacteria Using Mass Spectrometry. Anal. Chem. 1975, 47, 219–225.
Beverly, M. B.; Basile, F.; Voorhees, K. J.; Hadfield, T. L. A Rapid Approach for the Detection of Dipicolinic Acid in Bacterial Spores Using Pyrolysis Mass Spectrometry. Rapid Commun. Mass Spectrom. 1996, 10, 455–458.
Cain, T. C.; Lubman, D. M.; Weber, W. J. Differentiation of Bacteria Using Protein Profiles from Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1994, 8, 1026–1030.
Claydon, M. A.; Davey, S. N.; Edwards-Jones, V.; Gordon, D. B. The Rapid Identification of Intact Microorganisms Using Mass Spectrometry. Nat. Biotechnol. 1996, 14, 1584–1586.
Holland, R. D.; Wilkes, J. G.; Rafii, F.; Sutherland, J. B.; Persons, C. C.; Voorhees, K. J.; Lay, J. O. Rapid Identification of Intact Whole Bacteria Based on Spectral Patterns Using Matrix-Assisted Laser Desorption/Ionization with Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1996, 10, 1227–1232.
Krishnamurthy, T.; Ross, P. L.; Rajamani, U. Detection of Pathogenic and Non-Pathogenic Bacteria by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1996, 10, 883–888.
Krishnamurthy, T.; Ross, P. L. Rapid Identification of Bacteria by Direct Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Whole Cells. Rapid Commun. Mass Spectrom. 1996, 10, 1992–1996.
Williams, T. L.; Andrzejewski, D.; Lay, J. O.; Musser, S. M. Experimental Factors Affecting the Quality and Reproducibility of MALDI TOF Mass Spectra Obtained from Whole Bacteria Cells. J. Am. Soc. Mass Spectrom. 2003, 14, 342–351.
Setlow, P.; Johnson, E. A. Spores and Their Significance Doyle, M. P.; Beuchart, L. R.; Montville, T. J. Eds. Food Microbiology Fundamentals and Frontier; ASM Press: Washington DC., 1996, pp 30–65.
Beverly, M. B.; Voorhees, K. J.; Hadfield, T. L. Direct Mass Spectrometric Analysis of Bacillus Spores. Rapid Commun. Mass Spectrom. 1999, 13, 2320–2326.
Birmingham, J.; Demirev, P.; Ho, Y. P.; Thomas, J.; Bryden, W.; Fenselau, C. Corona Plasma Discharge for Rapid Analysis of Microorganisms by Mass Spectrometry. Rapid Commun. Mass Spectrom. 1999, 13, 604–606.
Elhanany, E.; Barak, R.; Fisher, M.; Kobiler, D.; Altboum, Z. Detection of Specific Bacillus Anthracis Spore Biomarkers by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 2001, 15, 2110–2116.
Hathout, Y.; Demirev, P. A.; Ho, Y. P.; Bundy, J. L.; Ryzhov, V.; Sapp, L.; Stutler, J.; Jackman, J.; Fenselau, C. Identification of Bacillus Spores by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Appl. Environ. Microbiol. 1999, 65, 4313–4319.
Ryzhov, V.; Hathout, Y.; Fenselau, C. Rapid Characterization of Spores of Bacillus Cereus Group Bacteria by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Appl. Environ. Microbiol. 2000, 66, 3828–3834.
Hathout, Y.; Setlow, B.; Cabrera-Martinez, R. M.; Fenselau, C.; Setlow, P. Small, Acid-Soluble Proteins as Biomarkers in Mass Spectrometry Analysis of Bacillus Spores. Appl. Environ. Microbiol. 2003, 69, 1100–1107.
Afonso, C.; Fenselau, C. Use of Bioactive Glass Slides for Matrix-Assisted Laser Desorption/Ionization Analysis: Application to Microorganisms. Anal. Chem. 2003, 75, 694–697.
Cazemier, A. E.; Wagenaars, S. F. M.; ter Steeg, P. F. Effect of Sporulation and Recovery edium on the Heat Resistance and Amount of Injury of Spores from Spoilage Bacilli. J. Appl. Microbiol. 2001, 90, 761–770.
Schaeffer, P. J.; Millet, J.; Aubert, J.-P. Catabolic Repression of Bacterial Sporulation. Proc. Natl. Acad. Sci. U.S.A. 1965, 54, 704–711.
Kooiman, W. J. The Screw Cap Tube Technique: A New and Accurate Technique for the Determination of the Wet Heat Resistance of Bacterial Spores. In Spore Research; Barker, A. N.; Gould, G. W.;, Wolf, J.; Eds.; Academic Press: London, 1973, pp 87–92.
Vorm, O.; Roepstorff, P.; Mann, M. Improved Resolution and Very High-Sensitivity in MALDI TOF of Matrix Surfaces Made by Fast Evaporation. Anal. Chem. 1994, 66, 3281–3287.
Strupat, K.; Karas, M.; Hillenkamp, F. 2,5-Dihydroxybenzoic Acid—A New Matrix for Laser Desorption/Ionization Mass Spectrometry. Int. J. Mass Spectrom. Ion Processes 1991, 111, 89–102.
Notz, R. E.; Haverkamp, J.; Horneffer, V., unpublished.
Demirev, P. A.; Ramirez, J.; Fenselau, C. Tandem Mass Spectrometry of Intact Proteins for Characterization of Biomarkers from Bacillus cereus T Spores. Anal. Chem. 2001, 73, 5725–5731.
Leuschner, R. G. K.; Lillford, P. J. Investigation of Bacterial Spore Structure by High Resolution Solid-State Nuclear Magnetic Resonance Spectroscopy and Transmission Electron Microscopy. Int. J. Food Microbiol. 2001, 63, 35–50.
Gould, G. W. Bacterial Endospores. In Encycopedia of Food Microbiology; Robinson, R. K.; Batt, C. A.; Patel, P. D., Eds.; Academic Press: London, 2000; pp 168–172.
Saenz, A. J.; Petersen, C. E.; Valentine, N. B.; Gantt, S. L.; Jarman, K. H.; Kingsley, M. T.; Wahl, K. L. Reproducibility of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Replicate Bacterial Culture Analysis. Rapid Commun. Mass Spectrom. 1999, 13, 1580–1585.
Evason, D. J.; Claydon, M. A.; Gordon, D. B. Effects of Ion Mode and Matrix Additives in the Identification of Bacteria by Intact Cell Mass Spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 669–672.
Madonna, A. J.; Basile, F.; Ferrer, I.; Meetani, M. A.; Rees, J. C.; Voorhees, K. J. On-Probe Sample Pretreatment for the Detection of Proteins Above 15 KDa from Whole Cell Bacteria by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 2220–2229.
Vaidyanathan, S.; Winder, C. L.; Wade, S. C.; Kell, D. B.; Goodacre, R. Sample Preparation in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Whole Bacterial Cells and the Detection of High Mass (>20 KDa) Proteins. Rapid Commun. Mass Spectrom. 2002, 16, 1276–1286.
Beavis, R. C.; Bridson, J. N. Epitaxial Protein Inclusion in Sinapic Acid Crystals. J. Phys. D Appl. Phys. 1993, 26, 442–447.
Horneffer, V.; Dreisewerd, K.; Lüdemann, H. C.; Hillenkamp, F.; Läge, M.; Strupat, K. Is the Incorporation of Analytes into Matrix Crystals a Prerequisite for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry? A Study of Five Positional Isomers of Dihydroxybenzoic Acid. Int. J. Mass Spectrom. 1999, 187, 859–870.
Horneffer, V.; Forsmann, A.; Strupat, K.; Hillenkamp, F.; Kubitscheck, U. Localization of Analyte Molecules in MALDI Preparations by Confocal Laser Scanning Microscopy. Anal. Chem. 2001, 73, 1016–1022.
Glückmann, M.; Pfenninger, A.; Krüger, R.; Thierolf, M.; Karas, M.; Horneffer, V.; Hillenkamp, F.; Strupat, K. Mechanisms in MALDI Analysis: Surface Interaction or Incorporation of Analytes?. Int. J. Mass Spectrom. 2001, 210, 121–132.
Andersson, A.; Ronner, U. Adhesion and Removal of Dormant, Heat-Activated, and Germinated Spores of Three Strains of Bacillus cereus. Biofouling 1998, 13, 51–67.
Peng, J. S.; Tsai, W. C.; Chou, C. C. Surface Characteristics of Bacillus cereus and Its Adhesion to Stainless Steel. Int. J. Food Microbiol. 2001, 65, 105–111.
Ryzhov, V.; Bundy, J. L.; Fenselau, C.; Taranenko, N.; Doroshenko, V.; Prasad, C. R. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Analysis of Bacillus Spores Using a 2.94 µm Infrared Laser. Rapid Commun. Mass Spectrom. 2000, 14, 1701–1706.
Beavis, R. C.; Chaudhary, T.; Chait, B. T. α-Cyano-4-hydroxycinnamic Acid as a Matrix for Matrix-Assisted Laser Desorption Mass Spectrometry. Org. Mass Spectrom. 1992, 27, 156–158.
Rosinke, B.; Strupat, K.; Hillenkamp, F.; Rosenbusch, J.; Dencher, N.; Krüger, U.; Galla, H. J. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) of Membrane-Proteins and Noncovalent Complexes. J. Mass Spectrom. 1995, 30, 1462–1468.
Zhu, Y. F.; Chung, C. N.; Taranenko, N. I.; Allman, S. L.; Martin, S. A.; Haff, L.; Chen, C. H. The Study of 2,3,4-Trihydroxyacetophenone and 2,4,6-Trihydroxyacetophenone as Matrices for DNA Detection in Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1996, 10, 383–388.
Stahl, B.; Steup, M.; Karas, M.; Hillenkamp, F. Analysis of Neutral Oligosaccharides by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Anal. Chem. 1991, 63, 1463–1466.
Karas, M.; Ehring, H.; Nordhoff, E.; Stahl, B.; Strupat, K.; Hillenkamp, F.; Grehl, M.; Krebs, B. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry with Additives to 2,5-Dihydroxybenzoic Acid. Org. Mass Spectrom. 1993, 28, 1476–1481.
Strupat, K.; Kampmeier, J.; Horneffer, V. Investigations of 2,5-DHB and Succinic Acid as Matrices for UV and IR MALDI. Part II. Crystallographic and Mass Spectrometric Analysis. Int. J. Mass Spectrom. 1997, 169, 43–50.
Horneffer, V.; Reichelt, R.; Strupat, K. Protein Incorporation into MALDI Matrix Crystals Investigated by High Resolution Field Emission Scanning Electron Microscopy. Int. J. Mass Spectrom. 2003, 226, 117–131.
Dai, Y. Q.; Whittal, R. M.; Li, L. Confocal Fluorescence Microscopic Imaging for Investigating the Analyte Distribution in MALDI Matrices. Anal. Chem. 1996, 68, 2494–2500.
Fournier, I.; Tabet, J. C.; Bolbach, G. Irradiation Effects in MALDI and Surface Modifications. Part I. Sinapinic Acid Monocrystals. Int. J. Mass Spectrom. 2002, 219, 515–523.
Ramirez, J.; Fenselau, C. Factors Contributing to Peak Broadening and Mass Accuracy in the Characterization of Intact Spores Using Matrix-Assisted Laser Desorption/Ionization Coupled with Time-of-Flight Mass Spectrometry. J. Mass Spectrom. 2001, 36, 929–936.
Amiri-Eliasi, B. J.; Fenselau, C. Characterization of Protein Biomarkers Desorbed by MALDI from Whole Fungal Cells. Anal. Chem. 2001, 73, 5228–5231.
Valentine, N. B.; Wahl, J. H.; Kingsley, M. T.; Wahl, K. L. Direct Surface Analysis of Fungal Species by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 2002, 16, 1352–1357.
Li, T. Y.; Liu, B. H.; Chen, Y. C. Characterization of Aspergillus Spores by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 2393–2400.
Welham, K. J.; Domin, M. A.; Johnson, K.; Jones, L.; Ashton, D. S. Characterization of Fungal Spores by Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 307–310.
Easterling, M. L.; Colangelo, C. M.; Scott, R. A.; Amster, I. J. Monitoring Protein Expression in Whole Bacterial Cells with MALDI Time-of-Flight Mass Spectrometry. Anal. Chem. 1998, 70, 2704–2709.
Domin, M. A.; Welham, K. J.; Ashton, D. S. The Effect of Solvent and Matrix Combinations on the Analysis of Bacteria by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1999, 13, 222–226.
Ryzhov, V.; Fenselau, C. Characterization of the Protein Subset Desorbed by MALDI from Whole Bacterial Cells. Anal. Chem. 2001, 73, 746–750.
Bright, J. J.; Claydon, M. A.; Soufian, M.; Gordon, D. B. Rapid Typing of Bacteria Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Pattern Recognition Software. J. Microbiol. Methods 2002, 48, 127–138.
Karas, M.; Glückmann, M.; Schäfer, J. Ionization in Matrix-Assisted Laser Desorption/Ionization: Singly Charged Molecular Ions are the Lucky Survivors. J. Mass Spectrom. 2000, 35, 1–12.
Knochenmuss, R.; Zenobi, R. MALDI Ionization: The Role of in-Plume Processes. Chem. Rev. 2003, 103, 441–452.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online September 11, 2004
Rights and permissions
About this article
Cite this article
Horneffer, V., Haverkamp, J., Janssen, HG. et al. MALDI-TOF-MS analysis of bacterial spores: Wet heat-treatment as a new releasing technique for biomarkers and the influence of different experimental parameters and microbiological handling. J Am Soc Mass Spectrom 15, 1444–1454 (2004). https://doi.org/10.1016/j.jasms.2004.06.017
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2004.06.017