Mass spectrometric strategy for the characterization of lipooligosaccharides from Neisseria gonorrhoeae 302 using FTICR

Abstract

The lipooligosaccharides (LOS) of Neisseria gonorrhoeae 302 were profiled using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Using techniques developed in this laboratory, the topology and some of the linkages of the LOS were determined. Mass spectrometric analysis in the negative ion mode yielded a glycoform of the composition: Hex 3 Hep 2 Hxn 1 PEA 1 KDO 2 DPLA. The composition was confirmed through exact mass measurements, which showed only a 2 ppm error between the exact mass and theoretical mass. Although the core structure has been postulated previously, the positioning of the three hexose moieties were in question for this particular strain of N. gonorrhoeae. Topology assignment was performed through collision-induced dissociation analysis of the O-deacylated glycoform in the negative ion mode followed by submission to the saccharide topology analysis tool (STAT) computer program, which confirmed the topology assignment. It was found that the three hexoses were added to the Hep[I] of the conserved core of N. gonorrhoeae in a linear fashion, while Hep[II] remains unbranched. Linkage position analysis was performed through application of a mild acid hydrolysis technique followed by collisioninduced dissociation of the sodiated precursor ions, yielding a 1 3 4 linkage between the terminating and penultimate hexoses. (J Am Soc Mass Spectrom 2002, 13, 571-576)

The Oligosaccharide of Gonococcal Lipooligosaccharide Contains Several Epitopes That Are Recognized by Human Antibodies

“…Gonococci were cultured on a GC agar base (Difco Laboratories, Detroit, MI) containing 1% defined supplement in a CO 2 incubator at 37°C (39,43). We used the following neisserial LOSs that have been immunochemically and/or structurally characterized: 2,3:3,4-dibranched LOSs (JW31R [5,42], 15253 [41], 15253 lgtE mutant [1], and WG [42]) and 3,4-branched LOSs (F62 [40], 24-1 [27], MS11mkA [18], PID-2 [38], 302 [21], and 220 [23]). LOS samples from proteinase K-digested (PK) cell lysates of gonococcal strains were prepared using the method of Hitchcock and Brown (13).…”

“…It only bound to each of their ϳ3.6-kDa minor components. The 220 LOS was recognized by MAb 2-1-L8 (23) (21).…”

Application of Fourier transform ion cyclotron resonance mass spectrometry to oligosaccharides

“…Their results suggested that the lipooligosaccharides extracts from Haemophilus influenza have extensive microhetrogeniety with structural isomers having different connectivities. As another application of FTICR, the structure of lipooligosaccharides from Neisseria gonorrhoeae 302 lipooligosaccharides has been reported (Leavell, Leary, & Yamasaki, 2002). With the mass accuracy of FTICR, the lipooligosaccharide composition was confirmed through exact mass measurements.…”

“…ESI-FTICR was used to characterize the structures of lipooligosaccharides Leavell, Leary, & Yamasaki, 2002). Sequence and linkage information of unknown lipooligosaccharide glycoforms were obtained from Haemophilus influenza 2019 using tandem MS (MS n ) analysis on FTICR and quadrupole ion trap instruments in combination with alkali metal ion coordination without permethylation or peracetylation .…”

The Biology of Neisseria Adhesins

“…A variety of biochemical and biophysical techniques have been used for LOS structural determination: these include methylation analysis, specific degradations (e.g., mild acid hydrolysis) and high-pH anion-exchange chromatography of underivatised oligosaccharides with reverse-phase high performance liquid chromatography; pulsed amperometric detection (HPAE-PAD) of oligosaccharides (OS), fluorophore-assisted carbohydrate electrophoresis monosaccharide composition analysis; 1D 500-MHz 1 H-NMR as well as nuclear Overhauser effect experiments; 2D NMR methods [double quantum filtered COSY (DQF-COSY), delayed COSY (D-COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA) and pure-absorption 2D NOE NMR]; mass spectrometry techniques (positive ion fast atom bombardment; Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS); electrospray ionization, collision-induced dissociation, and multiple step) [164,165,166,167,168,169,170,171,172,173,174]. Structural information on LOS has been recorded for meningococci [168,172,174,175,176,177], gonococci [178,179] and several commensals ( N. canis , N. perflava , N. subflava , N. flava , N. cinerea , N. flavescens , N. caviae , N. sicca ) [180,181,182].…”