Chapter 1
Rapid Characterization of Microorganisms by Mass Spectrometry: An Overview
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Catherine Fenselau1 and Plamen Demirev*,2 1Department
of Chemistry and Biochemistry, University of Maryland, College Park, MD 2Applied Physics Laboratory, Johns Hopkins University, Laurel, MD 20723 *
[email protected] Mass spectrometry approaches for rapid characterization of microorganisms date back more than thirty five years. Recent instrument and methods developments have brought to the fore new and exciting application in a number of fields, reviewed in individual chapters of this book.
This book contains chapters from participants in a recent symposium, organized by the Division of Analytical Chemistry during the ACS National Meeting in Washington DC in August, 2009, as well as several other active researchers in the field. The book covers aspects of mass spectrometry (MS) applications for microorganism characterization in several fields: biodefense, clinical diagnostics, food safety, environmental monitoring, and chemotaxonomy/biosystematics. The diverse list of contributors - from academia, government as well as industry – present multi-faceted and broad perspectives on the subject to be presented. Through the last thirty-five years MS has continuously provided profiles of chemical ensembles that are characteristic of different species of microorganisms. Such analyses have benefited from the strengths of mass spectrometry—speed, sensitivity, specificity and automation—and have also been influenced by its limitations, including costly MS instrumentation. The progress in mass spectrometry ionization methods and mass analyzers has been reflected in the particular approaches and protocols for microorganism analysis developed during this time. Although phospholipids and low mass metabolites were initially recognized as species specific biomarkers (1, 2), matrix-assisted © 2011 American Chemical Society In Rapid Characterization of Microorganisms by Mass Spectrometry; Fenselau, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.
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laser desorption/ionization (MALDI), electrospray and other advances in mass spectrometry have made it possible to profile and identify proteins and oligonucleotides (3) from bacteria. Two complementary strategies have evolved for assigning the identities of bacteria based on protein profiles detected in mass spectra. In one approach, careful culturing and controlled MALDI measurements allow the fingerprint comparison of a sample spectrum to a library of target reference spectra, and has been evaluated recently for use in clinical diagnostics (4). The other, a proteomic strategy, does not require a library of reference spectra, but uses bioinformatics (5, 6). Detected proteins are identified by relating suites of masses and partial sequences to a library of genomic sequences. Partial protein sequences can be obtained experimentally using standard bottom up proteomic techniques to identify peptides generated in bacterial lysates (7, 8) or from top down MS/MS analysis of intact proteins (9, 10). Recognizing biomarkers based on sequence tags allows reliable analysis of bacteria in mixtures (11) and characterization of bacteria that have been genetically engineered (12). Advances in bioinformatics have facilitated the identification of proteins in bacteria that lack a sequenced genome (13), and the phylogenetic characterization of such bacteria (14, 15). Several comprehensive reviews of the field (e.g., (3, 16, 17)) and two books (18, 19) have appeared recently. Here we list a number of issues that in our opinion are likely to influence future developments in the field: •
• • • • •
The first of these is the need for a standard spectral library that is independent of instrument and manufacturer. A mechanism should be provided for independent researchers to contribute spectra to this library. Signature validation and annotation will be required for applications in clinical and regulatory areas. Continued sequencing of microorganism genomes should be encouraged, along with contribution of annotated genomes to public databases. Readily achievable extensions should be explored, including the detection/analysis of drug resistance in bacteria. Time for analysis could be shortened considerably by new methods for analysis of mixtures. The community should aspire to develop robust, inexpensive, field-deployable systems that provide “end-to-end” capability, that is, systems that collect and prepare the sample, in addition to introducing it into the mass spectrometer and reporting out analysis of the data. Such systems are more likely to be adapted in hospitals and emergency units.
These and other developments will undoubtedly enhance the successful introduction of mass spectrometry in clinical microbiology and infectious disease diagnostics. In summary, we are pleased to bring together this set of current reports from leading contributors to the field, which we hope will stimulate further advances in both the technology and its applications.
2 In Rapid Characterization of Microorganisms by Mass Spectrometry; Fenselau, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.
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Pseudomonas putida strains by MALDI-MS using ribosomal subunit proteins as biomarkers. Anal. Chem. 2007, 79, 8712–8719. Wynne, C.; Edwards, N.; Fenselau, C. Phyloproteomic classification of unsequenced organisms by top-down identification of bacterial proteins using capLC-MS/MS on an Orbitrap. Proteomics 2010, 10, in press. Demirev, P.; Fenselau, C. Mass spectrometry in biodefense. J. Mass Spectrom. 2008, 43, 1441–1457. Ho, Y. P.; Reddy, P. M. Identification of pathogens by mass spectrometry. Clin. Chem. 2010, 56, 525–536. Identification of Microorganisms by Mass Spectrometry; Wilkins C. L., Lay, J. O., Eds.; Wiley-Interscience: New York, 2005. Mass Spectrometry for Microbial Proteomic; Shah, H. N., Gharbia, S. E., Eds.; Wiley-Interscience: New York, 2010.
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