Structural characterization of sialylated glycoforms of H. influenzae by electrospray mass spectrometry: fragmentation of protonated and sodiated O-deacylated lipopolysaccharides.

Sialylated lipopolysaccharide (LPS) glycoforms from Haemophilus influenzae were characterized by tandem mass spectrometry using a new generation hyphenated mass spectrometer which combines a triple quadrupole and a linear ion trap (Q-Trap). The fragmentation of both protonated and sodiated molecular ions from O-deacylated LPS (LPS-OH) obtained in MS(2) experiments in the positive mode was studied. The MS(2) spectra of protonated ions provided unambiguous evidence for the presence and sequence of sialylated lactosamine present in lacto-N-neotetraose oligosaccharide extensions but not for sialyl-lactose structures whilst fragmentation of sodiated adducts, [M+Na](+), afforded information diagnostic of mono- and disialylated lactose extensions. To study this we used a highly sialylated LPS from a H. influenzae strain capable of sialyl-lactose expression only. We then applied the method to the H. influenzae genome strain, Rd, in which glycoforms containing both sialyl-lactose and sialyl-lacto-N-neotetraose were detected from diagnostic B-ions at m/z 638.2 ([Neu5Ac(1) Hex(2)+Na](+)) and 657.2 ([Neu5Ac(1) Hex(1) HexNAc(1)+H](+)). Unique fragmentation patterns provided the locations and sequences of these oligosaccharide extensions. This is the first time both sialylated lactose and sialylated lacto-N-neotetraose units have been detected and characterized by tandem mass spectrometry in the same molecule. This methodology is of general applicability for determination of common sialylated oligosaccharide extension in bacterial LPS.

[1]  Jianjun Li,et al.  Identification of a Bifunctional Lipopolysaccharide Sialyltransferase in Haemophilus influenzae , 2006, Journal of Biological Chemistry.

[2]  Yutaka Akiyama,et al.  A computational study of structure-reactivity relationships in Na-adduct oligosaccharides in collision-induced dissociation reactions. , 2006, Carbohydrate research.

[3]  B. Gibson,et al.  Novel Sialic Acid Transporter of Haemophilus influenzae , 2005, Infection and Immunity.

[4]  J. Jurcisek,et al.  Role of Sialic Acid and Complex Carbohydrate Biosynthesis in Biofilm Formation by Nontypeable Haemophilus influenzae in the Chinchilla Middle Ear , 2005, Infection and Immunity.

[5]  D. Harvey Structural determination of N‐linked glycans by matrix‐assisted laser desorption/ionization and electrospray ionization mass spectrometry , 2005, Proteomics.

[6]  B. Domon,et al.  A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates , 1988, Glycoconjugate Journal.

[7]  Jianjun Li,et al.  Biosynthesis of Cryptic Lipopolysaccharide Glycoforms in Haemophilus influenzae Involves a Mechanism Similar to That Required for O-Antigen Synthesis , 2004, Journal of bacteriology.

[8]  Jianjun Li,et al.  Coupling capillary electrophoresis and high-field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of complex lipopolysaccharides. , 2004, Analytical chemistry.

[9]  B. Gibson,et al.  Nontypeable Haemophilus influenzae Strain 2019 Produces a Biofilm Containing N-Acetylneuraminic Acid That May Mimic Sialylated O-Linked Glycans , 2004, Infection and Immunity.

[10]  J. Brisson,et al.  Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Brisson,et al.  Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus influenzae. , 2002, European journal of biochemistry.

[12]  B. Gibson,et al.  Haemophilus influenzae Type b Strain A2 Has Multiple Sialyltransferases Involved in Lipooligosaccharide Sialylation* , 2002, The Journal of Biological Chemistry.

[13]  C. Lebrilla,et al.  Evidence for long-range glycosyl transfer reactions in the gas phase , 2002, Journal of the American Society for Mass Spectrometry.

[14]  R. Dwek,et al.  "Internal residue loss": rearrangements occurring during the fragmentation of carbohydrates derivatized at the reducing terminus. , 2002, Analytical chemistry.

[15]  W. Wakarchuk,et al.  Genetic basis for expression of the major globotetraose-containing lipopolysaccharide from H. influenzae strain Rd (RM118). , 2001, Glycobiology.

[16]  D. Hood,et al.  A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae: a survey of 24 non-typeable H. influenzae strains. , 2001, Carbohydrate research.

[17]  J. Brisson,et al.  Identification of a lipopolysaccharide α‐2,3‐sialyltransferase from Haemophilus influenzae , 2001, Molecular microbiology.

[18]  Structure and functional genomics of lipopolysaccharide expression in Haemophilus influenzae. , 2001, Advances in experimental medicine and biology.

[19]  J. Leary,et al.  Mass spectral characterization of lipooligosaccharides from Haemophilus influenzae 2019. , 2000, Biochemistry.

[20]  E. Vimr,et al.  Sialic acid metabolism's dual function in Haemophilus influenzae , 2000, Molecular microbiology.

[21]  P. Thibault,et al.  Sialic acid in the lipopolysaccharide of Haemophilus influenzae: strain distribution, influence on serum resistance and structural characterization , 1999, Molecular microbiology.

[22]  D. Hood,et al.  Haemophilus influenzae lipopolysaccharide. , 1999, Biochemical Society transactions.

[23]  E. Moxon,et al.  Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by a capsule-deficient strain of Haemophilus influenzae Rd. , 1999, European journal of biochemistry.

[24]  J. Thomas-Oates,et al.  Sodium-cationized oligosaccharides do not appear to undergo 'internal residue loss' rearrangement processes on tandem mass spectrometry. , 1998, Rapid communications in mass spectrometry : RCM.

[25]  J. Brisson,et al.  Structure of an alpha-2,6-sialylated lipooligosaccharide from Neisseria meningitidis immunotype L1. , 1998, European journal of biochemistry.

[26]  C. Lebrilla,et al.  Effects of cations and charge types on the metastable decay rates of oligosaccharides. , 1994, Analytical chemistry.

[27]  M. Apicella,et al.  Lipo-oligosaccharides (LOS) of mucosal pathogens: molecular mimicry and host-modification of LOS. , 1993, Immunobiology.

[28]  M. Wilson,et al.  Modification by sialic acid of Neisseria gonorrhoeae lipooligosaccharide epitope expression in human urethral exudates: an immunoelectron microscopic analysis. , 1990, The Journal of infectious diseases.