Introduction Recent research has proposed rapid and robust identification of intact microorganisms using matrix assisted laser desorption/ ionization time-of-flight mass spectrometry and bioinformatics [1]. Previous work has relied primarily on desorption and detection of protein biomarkers weighing less than 20 kilo Daltons. Analysis of prokaryotic genomes predicts fewer proteins with higher masses per organism and, thus, the potential to provide more definitive microorganism identifications. However, higher mass proteins have not yet been readily accessible by MALDI and widely evaluated for rapid detection of bacteria. They are difficult to desorb because of suppression by other components of the lysed cell; they are detected with less sensitivity by most commercial ion detectors. This poster reports a procedure using acid-cleavable detergent and microwave to facilitate desorption of higher molecular weight protein biomarkers from lysed whole cells. This approach has been evaluated with Escherichia coli (K-12), Salmonella typhimurium, Bacillus anthracis Sterne, and Bacillus subtilis (168). A microwave and detergent procedure to detect high molecular mass proteins from vegetative bacteria by MALDI-TOF MS Elizabeth Patton 1 ; Nathan Edwards 2 ; Berk Oktem 3 ; and Catherine Fenselau 1 1 Chemistry and Biochemistry and 2 Center for Bioinformatics, University of Maryland, College Park, MD; 3 Middle Atlantic Mass Spectrometry Lab, Johns Hopkins School of Medicine, Baltimore, MD Literature cited 1.Fenselau, C.; Demirev, P. A. Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrom. Rev. 2001, 20, Norris, J. L.; Porter, N. A.; Caprioli, R. M. Mass spectrometry of intracellular and membrane proteins using cleavable detergents. Analytical Chemistry 2003, 75, Wessel, D.; Flugge, U. I. A Method for the Quantitative Recovery of Protein in Dilute- Solution in the Presence of Detergents and Lipids. Analytical Biochemistry 1984, 138, Experimental methods and materials Intact Cells Disrupt cell membrane and solubilize proteins with acid cleavable detergent Clean up protein with a Folch extraction [3] Mix protein with saturated sinapinic acid (SA) Lower pH and microwave Sample applied on top of dried layer of saturated SA crystals on slide MALDI-TOF instruments: Kratos Axima CFR+ and Comet Macromizer 20kDa 100kD Search Rapid Microorganism Identification DataBase for best match Zwitterionic 6-PPS Detergent [2] Enhances the solubility of hydrophobic proteins Acid cleavable by microwaving at pH 1.4 Eliminates detergent signal interference Solvent System [3]  Salts  protein fraction at the interface  Lipids C 22 H 39 NO 5 S CHCl 3 CH 3 OH H 2 O Objectives 1.To evaluate accessibility by MALDI to higher mass proteins in intact bacteria 2.To evaluate the suitability of higher mass proteins to provide identifications based on genomic database searching Results %Int Mass/Charge %Int Mass/Charge %Int Mass/Charge %Int Mass/Charge Escherichia coli Salmonella typhimurium Bacillus subtilis 168 Bacillus anthracis Sterne Escherichia coli Salmonella typhimurium Salmonella typhimurium Bacillus subtilis Bacillus anthracis Sterne Reproducibility Conclusion This procedure afforded the desorption and detection of several proteins from intact species in molecular mass ranges above 20kDa. However, good spectra with peaks in the 20-50kDa range aren't enough for microorganism identification. To make high-mass biomarkers suitable requires one or more of the following advances, all of which increase the specificity of a spectrum peak with respect to its species/organism. a) a significant boost in mass accuracy, b) an understanding of which proteins, or protein subset, have abundant peaks in these spectra, c) obtaining peaks in a mass range above 80kDa. Comparison of Axima spectra of four species Comparison of Comet Macromizer spectra of four species The cryodetector is more sensitive to higher masses because the signal is independent of mass and, thus, impact velocity. Spectra are reproducible from spot to spot. Escherichia coli This simulation illustrates that high-mass, in the 20-50KDa range, is insufficient for microorganism identification with current technologies. Ribosomal proteins from the RMIDB database were selected and then a random error, based on the instrument accuracy (x-axis), was applied to their exact theoretical masses; the RMIDB was used to "look-up" the spectrum (number of peaks detected on y-axis) and check the e-value (z-axis) of the correct answer.