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A microwave and detergent procedure to detect high molecular mass proteins from vegetative bacteria by MALDI-TOF MS Elizabeth Patton1; Nathan Edwards2; Berk Oktem3; and Catherine Fenselau1 Chemistry and Biochemistry and 2Center for Bioinformatics, University of Maryland, College Park, MD; 3 Middle Atlantic Mass Spectrometry Lab, Johns Hopkins School of Medicine, Baltimore, MD %Int. %Int. 90 Escherichia coli 80 Escherichia coli 70 35205 57335 55165 56159 59496 Salmonella typhimurium 80 50510 20 90 52106 35540 100 47873 40 %Int. 45793 46624 60000 43285 44097 55000 41287 50000 37389 38275 39147 45000 35418 40000 Mass/Charge 32388 33416 34421 35000 30348 31191 30000 28514 25000 25905 26665 20000 24654 60 0 20226 21198 22140 22991 59496 56159 80 57335 55165 52106 50510 47873 45793 10 46624 44097 41287 100 42682 38275 37389 39147 43285 35614 34421 32388 33416 30348 28514 20 31191 25905 26665 24654 25617 24227 30 Disrupt cell membrane and solubilize proteins with acid cleavable detergent Intact Cells 20887 40 22313 20226 50 22991 35418 60 0 20000 25000 30000 35000 40000 45000 50000 55000 60000 Mass/Charge 70 60 35751 50 45533 36867 37925 43289 35950 34664 32578 33487 31161 28424 10 29755 24658 26687 Zwitterionic 6-PPS Detergent [2] 25920 20 22325 20247 30 23010 40 0 20000 25000 30000 35000 40000 45000 50000 55000 60000 Mass/Charge 20703 46640 58888 59760 0.7 50653 47797 49199 46479 10 O 0.8 43542 42840 44286 39786 38200 37525 20 36862 36156 34153 35252 32883 31212 27725 29023 30 0.9 33710 27404 25764 24250 40 N 32682 22418 50 + 54713 41336 22760 60 Bacillus subtilis 168 1 30468 O 70 24917 O 80 23026 O- 90 21741 S H3C 100 20212 O Clean up protein with a Folch extraction [3] Mix protein with saturated sinapinic acid (SA) Lower pH and microwave %Int. 20032 CH3 0 20000 25000 30000 35000 40000 45000 50000 55000 60000 21116 %Int. 100 21905 Mass/Charge CH3 90 80 C22H39NO5S 24008 44113 30226 26624 25517 24693 20 10 0 0 20000 25000 30000 35000 40000 45000 50000 55000 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. 43334 37515 38049 38420 35316 17714 15553 Escherichia coli Salmonella typhimurium Salmonella typhimurium 91406 77979 Salts 103682 100kD 50918 43243 20kDa protein fraction at the interface Lipids Bacillus subtilis 168 Bacillus anthracis Sterne 23972 21063 21785 127445 89082 83965 43000 36082 163864 126474 Search Rapid Microorganism Identification DataBase for best match 112667 116000 CHCl3 3 5 10 8 peaks Solvent System [3] H2O 60000 Mass/Charge MALDI-TOF instruments: Kratos Axima CFR+ and Comet Macromizer CH3OH 50 30 20 ppm 15 0.1 36273 20293 21628 30 22972 21317 0.2 35493 • 40 37670 • Enhances the solubility of hydrophobic proteins Acid cleavable by microwaving at pH 1.4 Eliminates detergent signal interference 50 100838 • 0.3 60 Sample applied on top of dried layer of saturated SA crystals on slide 0.6 chance m atch 0.5 probability 0.4 Bacillus anthracis Sterne 70 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 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 DiluteSolution in the Presence of Detergents and Lipids. Analytical Biochemistry 1984, 138, 141-143. Spectra are reproducible from spot to spot. Comparison of Axima spectra of four species 100 Objectives Literature cited Reproducibility Results 54793 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). Experimental methods and materials 35205 Introduction 46349 1 10 13 5 15 20 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. 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.