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Standardization of Sample Preparation Techniques Used in Analyzing Microorganisms by MALDI-TOF Mass Spectrometry

Chad P. Nelson1, Barbara J. Porter1, Bryant C. Nelson1, Gary W. Mallard2, Edward White V1, Michael J. Welch1

1Analytical Chemistry Division and 2Physical and Chemical Properties Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD 20899

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has in recent years been utilized as a technique for fingerprinting and identifying microorganisms, including bacteria, viruses, fungi and even higher order eukaryotic cells.  Many efforts have focused on ways to produce unique mass spectra that can distinguish bacterial cells even at the strain level.  Although a number of successes have been achieved in this area, the means by which these bacterial mass fingerprint libraries have been derived are based on a wide number of different sample handling techniques that can drastically affect the observed fingerprint.  This field of research takes on added urgency with the need for rapid, definitive means of identifying bioterrorism agents as well as the need for credible, verifiable methods for the identification of organisms that may be covered by the Biological Weapons Convention.  In an effort to create a library of MALDI-TOF mass spectra for a large number of microorganisms and develop algorithms for distinguishing the spectra of different organisms and identifying known organisms, sample preparation procedures used in generating these unique spectra must first be evaluated and standardized.  We have tested a number of sample handling techniques including whole cell analysis, ultrasonication/lysis of cells, and extraction of proteins from cells using a variety of solvent systems.  Different wash solvents, MALDI matrices, combined MALDI matrices, and sample to matrix ratios were also evaluated.  MALDI-TOF spectra showed the greatest variability between different matrices.  Disruption of cells by ultrasonication or extraction of proteins with certain solvents produced fingerprints that were very similar to whole cell analysis, with the exception of several higher molecular weight ions between 20 kDa to 40 kDa present in the former.  Data collected on different samples of Eschericia coli indicated the presence of common biomarkers among the strains tested, as well as markers that may be specific to each strain. Standardization of sample preparation protocols and the development of mass spectral libraries and search algorithms to identify microorganisms, may aid in supporting the Biological Weapons Convention and may have wider implications for bacterial identification in other areas (e.g., environmental, biomedical research, clinical, food industry).