Direct Microextraction and Analysis of Rough-Type Lipopolysaccharides by Combined Thin-Layer Chromatography and MALDI Mass Spectrometry He ´le `ne Therisod, Vale ´rie Labas, and Martine Caroff* Equipe “Endotoxines”, UMR 8619 du Centre National de la Recherche Scientifique, Biochimie, Universite ´ de Paris-Sud, F-91405 Orsay, France A rapid method for the microscale extraction of li- popolysaccharides (endotoxins, LPSs) from rough-type Gram-negative bacteria was developed using thin-layer chromatography (TLC) combined with improved condi- tions for LPS analysis by mass spectrometry. TLC of intact bacteria on silica gel plates in an appropriate solvent selectively extracted and separated their LPS components. The bands of molecular species were scraped from the plates after nondestructive visualization, directly mixed with matrix, and analyzed by laser desorption time-of-flight mass spectrometry. Lipids A and Re-type LPSs were analyzed after transfer to a membrane. Adding citric acid to the matrix gave greatly improved mass spectra. The system allows characterization of bacterial LPS at the microscale level and is equally well applicable to hetero- geneous LPS and lipid A preparations (Escherichia coli lipid A and Bordetella lipopolysaccharides were used). The technique provides a rapid determination of the heterogeneity of unmodified preparations and the deter- mination of the molecular weight of each separated component. Endotoxins are powerful immunomodulators and the major components of Gram-negative bacterial outer membranes. They are mixtures of lipopolysaccharides (LPSs). The lipid domain of LPS, called lipid A, is responsible for most of the biological activities. In smooth-form LPS, it is linked through a core oligosaccharide to an O-chain carrying antigenic determinants. LPSs lacking O-chains are designated rough-type. Lipopolysaccharide and lipid A preparations are usually het- erogeneous. This can be evidenced on SDS electrophoretic gels 1 and by thin-layer chromatography 2,3 (TLC). Analysis and comparison of the separated structures are usually performed on samples from preparative-scale extraction. 4,5 How- ever, most commonly used methods for extracting both smooth- or rough-type LPSs are time-consuming and require an appreciable amount of material. Profiling of lysed bacteria introduced into a fast atom bombard- ment mass spectrometer 6 (FAB-MS) allowed detection and com- parison of membrane lipids. Plasma desorption mass spectrometry (PDMS) of native rough-type LPS was shown to be facilitated by decationization and disruption of micelles in plasma desorption mass spectrometry. 7 It was then found that native rough-type 8-10 and smooth-type 11 LPS mass spectra could be obtained with the matrix-assisted laser desorption/ ionization (MALDI) method. Recent results have shown that TLC-separated glycosphingolipids can be analyzed by introducing the plate directly into the mass spectrometer or by transferring the products to a membrane prior to MALDI mass spectrometry (MALDI-MS) analysis. 12 We present here a method for the direct selective extraction and separation of LPSs from bacterial cells, nondestructive visualization of the material on the plate, and transfer of the relevant parts of the silica gel to the mass spectrometer. The method can also be used for the direct MS analysis of individual TLC-separated components of lipid A and LPS preparations. New conditions for improving LPS analysis by MALDI-MS are also recorded. EXPERIMENTAL SECTION Bacterial Strains. Cells of Bordetella pertussis 1414 ( B. pertussis 1, wild type) and A100 ( B. pertussis 2, LPS mutant strain) were obtained as previously described. 13 Escherichia coli O119 strain 19392, Bordetella bronchiseptica 4098 and Bordetella para- pertussis strain 15989 were from the NRCC collection. 14 LPS. Cells were grown as described earlier. 14 The cells were killed in 2%phenol before harvesting. LPS was extracted by the * Corresponding author: (tel) 33 1 69 15 71 91; (fax) 33 1 69 85 37 15; (e-mail) Martine.Caroff@ bbmpc.u-psud.fr. (1) Peterson, A. A.; Haug, A.; McGroarty, E. J. J. Bacteriol. 1986 , 165, 116- 122. (2) Novotny, A. Nature 1966 , 210, 278-280. (3) Caroff, M.; Karibian, D. Appl. Environ. Microbiol. 1990 , 56, 1957-1959. (4) Lebbar, S.; Karibian, D.; Deprun, C.; Caroff, M. J. Biol. Chem. 1994 , 269, 31881-31884. (5) Zhou, Z.; Lin, S.; Cotter, R. J.; Raetz C. R. H. J. Biol. Chem. 1999 , 274, 18503-18514. (6) Heller, D. N.; Cotter, R. J.; Fenseleau, C.; Uy, O. M. Anal. Chem. 1987 , 59, 2806-2809. (7) Caroff, M.; Deprun, C.; Karibian, D. J. Biol. Chem. 1993 , 268, 12321-12324. (8) Gibson, B. W.; Engstrom, J. J.; John, C. M.; Hines, W.; Falick, A. M. J. Am. Soc. Mass. Spectrom. 1997 , 8, 645-658. (9) Fukuoka, S.; Knirel, Y. A.; Lindner, B.; Moll, H.; Seydel, U.; Za ¨ hringer, U. Eur. J. Biochem. 1997 , 250, 55-62. (10) Caroff, M.; Brisson, J. R.; Martin, A.; Karibian, D. FEBS Lett. 2000 , 477, 8-14. (11) Aussel, L.; Chaby, R.; Le Blay, K.; Kelly, J.; Thibault, P.; Perry, M. B.; Caroff, M. FEBS Lett. 2000 , 485, 40-46. (12) Guittard, J.; Hronowski, X.; Costello, C. E. Rapid Commun. Mass Spectrom. 1999 , 13 ( 18) , 1838-1849. (13) Caroff, M.; Chaby, R.; Karibian, D.; Perry, J.; Deprun, C.; Szabo, L. J. Bacteriol. 1990 , 172, 1121-1128. (14) Di Fabio; J. L., Caroff, M.; Karibian, D.; Richards, J. C.; Perry, M. B. FEMS Microbiol. Lett. 1992 . 76, 275-281. Anal. Chem. 2001, 73, 3804-3807 3804 Analytical Chemistry, Vol. 73, No. 16, August 15, 2001 10.1021/ac010313s CCC: $20.00 © 2001 American Chemical Society Published on Web 06/30/2001