1. 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, 157-171. 2.Norris, J. L.; Porter, N. A.; Caprioli, R. M. Mass spectrometry of intracellular and membrane proteins using cleavable detergents. Analytical Chemistry 2003, 75, 6642-6647. 3.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, 141-143. 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 0 10 20 30 40 50 60 70 80 90 100 %Int. 200002500030000350004000045000500005500060000 Mass/Charge 35205 35418 22991 20226 2590522313 24654 35614 20887 26665 43285 32388 33416 28514 25617 24227 34421 30348 31191 38275 44097 37389 45793 39147 47873 41287 42682 50510 52106 46624 56159 5516557335 59496 0 10 20 30 40 50 60 70 80 90 100 %Int. 200002500030000350004000045000500005500060000 Mass/Charge 35540 35751 23010 20247 35950 22325 24658 25920 43289 26687 31161 28424 29755 34664 3257837925 36867 33487 45533 0 10 20 30 40 50 60 70 80 90 100 %Int. 200002500030000350004000045000500005500060000 Mass/Charge 20703 41336 22760 54713 22418 20212 32682 20032 23026 27404 21741 33710 24250 25764 46640 32883 27725 29023 24917 35252 31212 34153 30468 43542 38200 39786 36156 37525 36862 42840 46479 44286 47797 50653 49199 59760 58888 0 10 20 30 40 50 60 70 80 90 100 %Int. 200002500030000350004000045000500005500060000 Mass/Charge 21905 21116 21317 24008 20293 21628 22972 36273 44113 24693 30226 2551726624 Escherichia coli Salmonella typhimurium Bacillus subtilis 168 Bacillus anthracis Sterne Escherichia coli 15553 17714 35316 37515 38049 38420 43334 Salmonella typhimurium 35493 37670 43243 50918 Salmonella typhimurium 77979 91406 103682 Bacillus subtilis 168 163864 126474 112667 116000 100838 54793 46349 127445 83965 89082 43000 36082 23972 21063 21785 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.