Abstract
We described a multiple-stage ion-trap mass spectrometric approach to characterize the structures of phosphatidylinositol and phosphatidyl-myoinositol mannosides (PIMs) in a complex mixture isolated from Mycobacterium bovis Bacillus Calmette Guérin. The positions of the fatty acyl substituents of PIMs at the glycerol backbone can be easily assigned, based on the findings that the ions arising from losses of the fatty acid substituent at sn-2 as molecules of acid and of ketene, respectively (that is, the [M − H − R2CO2H]− and [M − H − R2CH=CO]− ions), are respectively more abundant than the ions arising from the analogous losses at sn-1 (that is, the [M − H − R1CO2H]− and [M − H − R1CH=CO]− ions) in the MS2 product-ion spectra of the [M − H]− ions desorbed by electrospray ionization (ESI). Further dissociation of the [M − H − R2CO2H]− and [M − H − R1CO2H]− ions gives rise to a pair of unique ions corresponding to losses of 74 and 56 Da (that is, [M − H − RxCO2H − 56]− and [M − H − RxCO2H − 74]− ions, x=1, 2), respectively, probably arising from various losses of the glycerol. The profile of the ion-pair in the MS3 spectrum of the [M − H − R2CO2H]− ion is readily distinguishable from that in the MS3 spectrum of the [M − H − R1CO2H]− ion and thus the assignment of the fatty acid substituents at the glycerol backbone can be confirmed. The product-ion spectra of the [M − H]− ions from 2-lyso-PIM and from 1-lyso-PIM are discernible and both spectra contain a unique ion that arises from primary loss of the fatty acid substituent at the glycerol backbone, followed by loss of a bicyclic glycerophosphate ester moiety of 136 Da. The combined structural information from the MS2 and MS3 product-ion spectra permit the complex structures of PIMs that consist of various isomers to be unveiled in detail.
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Anderson, R. J.; Renfrew, A. G. The Chemistry of the Lipoids of Tubercle Bacilli: XIII. The Occurrence of Mannose in the Phosphatide from Human Tubercle Bacilli. J. Am. Chem. Soc. 1930, 52, 1252–1254.
Anderson, R. J. The Chemistry of the Lipoids of Tubercle Bacilli: XIV. The Occurrence of Inosite in the Phosphatide of Human Tubercle Bacilli. J. Am. Chem. Soc. 1930, 52, 1607–1608.
Anderson, R. J.; Roberts, E. G. The Chemistry of the Lipoids of Tubercle Bacilli: XXI. The Polysaccharide Occurring in the Phosphatide from the Human Tubercle Bacilli. J. Am. Chem. Soc. 1930, 52, 5023–5029.
Anderson, R. J.; Lothrop, W. C.; Creighton, M. M. The Chemistry of the Lipids of Tubercle Bacilli: LIII. Studies on the Phosphatide of the Human Tubercle Bacillus. J. Biol. Chem. 1938, 125, 299–308.
Lee, Y. C.; Ballou, C. E. Complete Structures of the Glycophospholipids of Mycobacteria. Biochemistry. 1965, 4, 1395–1404.
Ballou, C. E.; Vilkas, E.; Lederer, E. Structural Studies on the Myo-inositol Phospholipids of Mycobacterium tuberculosis (var. bovis, strain BCG). J. Biol. Chem. 1963, 238, 69–76.
Ballou, C. E.; Lee, Y. C. The Structure of a Myoinositol Mannoside from Mycobacterium tuberculosis Glycolipid. Biochemistry. 1964, 3, 682–685.
Lee, Y. C.; Ballou, C. E. Structural Studies on the Myoinositol Mannosides from the Glycolipids of Mycobacterium tuberculosis and Mycobacterium phlei. J. Biol. Chem. 1964, 239, 1316–1327.
Sasaki, A. Isolation and Characterization of Serologically Active Phosphatidylinositol Oligomannosides of Mycobacterium tuberculosis. J. Biochem. (Tokyo). 1975, 78, 547–554.
Hunter, S. W.; Brennan, P. J. Evidence for the Presence of a Phosphatidylinositol Anchor on the Lipoarabinomannan and Lipomannan of Mycobacterium tuberculosis. J. Biol. Chem. 1990, 265, 9272–9279.
Rhoades, E. R.; Hsu, F. F.; Torrelles, J. B.; Turk, J.; Chatterjee, D.; Russell, D. G. Identification and Macrophage-activating Activity of Glycolipids Released from Intracellular Mycobacterium bovis BCG. Mol. Microbiol. 2003, 48, 875–888.
Fischer, K.; Scotet, E.; Niemeyer, M.; Koebernick, H.; Zerrahn, J.; Maillet, S.; Hurwitz, R.; Kursar, M.; Bonneville, M.; Kaufmann, S. H. E.; Schaible, U. E. Mycobacterial Phosphatidylinositol Mannoside Is a Natural Antigen for CD1d-restricted T Cells. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10685–10690.
Chatterjee, D.; Khoo, K. H. Mycobacterial Lipoarabinomannan: An Extraordinary Lipoheteroglycan with Profound Physiological Effects. Glycobiology. 1998, 8, 113–120.
Apostolou, I.; Takahama, Y.; Belmant, C.; Kawano, T.; Huerre, M.; Marchal, G.; Cui, J.; Taniguchi, M.; Nakauchi, H.; Fournie, J. J.; Kourilsky, P.; Gachelin, G. Murine Natural Killer Cells Contribute to the Granulomatous Reaction Caused by Mycobacterial Cell Walls. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5141–5146.
Cywes, C.; Hoppe, H. C.; Daffe, M.; Ehlers, M. R. Nonopsonic Binding of Mycobacterium tuberculosis to Complement Receptor Type 3 Is Mediated by Capsular Polysaccharides and Is Strain Dependent. Infect. Immun. 1997, 65, 4258–4266.
Hoppe, H. C.; de Wet, B. J.; Cywes, C.; Daffe, M.; Ehlers, M. R. Identification of Phosphatidylinositol Mannoside as a Mycobacterial Adhesin Mediating Both Direct and Opsonic Binding to Nonphagocytic Mammalian Cells. Infect. Immun. 1997, 65, 3896–3905.
Beatty, W. L.; Rhoades, E. R.; Ullrich, H. J.; Chatterjee, D.; Heuser, J. E.; Russell, D. G. Trafficking and Release of Mycobacterial Lipids from Infected Macrophages. Traffic. 2000, 1, 235–247.
Chatterjee, D.; Hunter, S. W.; McNeil, M.; Brennan, P. J. Lipoarabinomannan: Multiglycosylated Form of the Mycobacterial Mannosylphosphatidylinositols. J. Biol. Chem. 1992, 267, 6228–6233.
Severn, W. B.; Furneaux, R. H.; Falshaw, R.; Atkinson, P. H. Chemical and Spectroscopic Characterisation of the Phosphatidylinositol Manno-oligosaccharides from Mycobacterium bovis AN5 and WAg201 and Mycobacterium smegmatis mc2 155. Carbohydr. Res. 1998, 308, 397–408.
Pangborn, M. C.; McKinney, J. A. Purification of Serologically Active Phosphoinositides of Mycobacterium tuberculosis. J. Lipid Res. 1966, 7, 627–633.
Brennan, P. J.; Ballou, C. E. Biosynthesis of Mannophosphoinositides by Mycobacterium phlei: Enzymatic Acylation of the Dimannophosphoinositides. J. Biol. Chem. 1968, 243, 2975–2984.
Khuller, G. K.; Subrahmanyam, D. On the Mannophosphoinositides of Mycobacterium 607. Experientia. 1968, 24, 851–852.
Khoo, K. H.; Dell, A.; Morris, H. R.; Brennan, P. J.; Chatterjee, D. Structural Definition of Acylated Phosphatidylinositol Mannosides from Mycobacterium tuberculosis: Definition of a Common Anchor for Lipomannan and Lipoarabinomannan. Glycobiology. 1995, 5, 117–127.
Gilleron, M.; Bala, L.; Brando, T.; Vercellone, A.; Puzo, G. Mycobacterium tuberculosis H37Rv Parietal and Cellular Lipoarabinomannans: Characterization of the Acyl- and Glyco-forms. J. Biol. Chem. 2000, 275, 677–684.
Gilleron, M.; Ronet, C.; Mempel, M.; Monsarrat, B.; Gachelin, G.; Puzo, G. Acylation State of the Phosphatidylinositol Monnosides from Mycobacterium bovis Bacillus Calmette Guérin and Ability to Induce Granuloma and Recruit Natural Killer T Cells. J. Biol. Chem. 2001, 276, 34896–34904.
Gilleron, M.; Quesniaux, V. F. J.; Puzo, G. Acylation State of the Phosphatidylinositol Hexammanosides from Mycobacterium bovis Bacillus Calmette Guérin and Mycobaterium tuberculosis H37Rv and Its Implication in toll-like receptor response. J. Biol. Chem. 2003, 278, 29880–29889.
Nigou, J.; Gilleron, M.; Brando, T.; Puzo, G. Structural Analysis of Mycobacterial Lipoglycans. Appl. Biochem. Biotechnol. 2004, 118, 253–268.
Hsu, F. F.; Turk, J.; Owens, R.; Rhoades, E. R.; Russell, D. G. Structural Characterization of Phosphatidylinositol Mannosides from Mycobacterium bovis BCG by Multiple-Stage Quadrupole Ion-Trap Mass Spectrometry with Electrospray Ionization. II. Monoacyl-and Diacyl-PIMs. J. Am. Soc. Mass Spectrom. 2007, 18, 479–492.
Folch, J.; Lees, M.; Sloane-Stanley, G. H. A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues. J. Biol. Chem. 1957, 226, 497–509.
Morita, Y. S.; Patterson, J. H.; Billman-Jasobe, H.; McConville, M. A. Biosynthesis of Mycobacterial Phosphatidylinositol Mannosides. Biochem. J. 2004, 378, 591–597.
Hsu, F. F.; Turk, J. Electrospray Ionization with Low-energy Collisionally Activated Dissociation Tandem Mass Spectrometry of Complex Lipids: Structural Characterization and Mechanisms of Fragmentation. In Modern Methods for Lipid Analysis; Byrdwell, W. C., ed.; AOCS Press: Champaign, IL, 2005, p. 61.
Hsu, F. F.; Turk, J. Characterization of Phosphatidylinositol, Phosphatidylinositol-4-phosphate, and Phosphatidylinositol-4,5-bisphosphate by Electrospray Ionization Tandem Mass Spectrometry: A Mechanistic Study. J. Am. Soc. Mass Spectrom. 2000, 11, 986–999.
Hsu, F. F.; Turk, J. Structural Determination of Glycosphingolipids as Lithiated Adducts by Electrospray Ionization Mass Spectrometry Using Low Energy Collisional-activated Dissociation on a Triple Stage Quadrupole Instrument. J. Am. Soc. Mass Spectrom. 2001, 12, 61–79.
Hsu, F. F.; Turk, J.; Rhoades, E. R.; Russell, D. G.; Shi, X.; Groisman, E. A. Structural Characterization of Cardiolipin by Tandem Quadrupole and Multiple-Stage Quadrupole Ion-Trap Mass Spectrometry with Electrospray Ionization. J. Am. Soc. Mass Spectrom. 2005, 16, 491–504.
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Published online November 30, 2006
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Hsu, FF., Turk, J., Owens, R.M. et al. Structural characterization of phosphatidyl-myo-inositol mannosides from Mycobacterium bovis bacillus calmette guérin by multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization. I. PIMs and lyso-PIMs. J Am Soc Mass Spectrom 18, 466–478 (2007). https://doi.org/10.1016/j.jasms.2006.10.012
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DOI: https://doi.org/10.1016/j.jasms.2006.10.012