Glycomics, the study of microbial polysaccharides and genes responsible for their formation, requires the continuous development of rapid and sensitive methods for the identification of glycan structures. In this study, methods for the direct analysis of sugars from 108 to 1010 cells are outlined using the human gastrointestinal pathogen, Campylobacter jejuni. Using capillary-electrophoresis coupled with sensitive electrospray mass spectrometry, we demonstrate variability in the lipid A component of C. jejuni lipooligosaccharides (LOSs). In addition, these sensitive methods have permitted the detection of phase-variable LOS core structures that were not observed previously. High resolution magic angle spinning (HR-MAS) NMR was used to examine capsular polysaccharides directly from campylobacter cells and showed profiles similar to those observed for purified polysaccharides analyzed by solution NMR. This method also exhibited the feasibility of campylobacter serotyping, mutant verification, and preliminary sugar analysis. HR-MAS NMR examination of growth from individual colonies of C. jejuni NCTC11168 indicated that the capsular glycan modifications are also phase-variable. These variants show different staining patterns on deoxycholate-PAGE and reactivity with immune sera. One of the identified modifications was a novel -OP=O(NH2)OMe phosphoramide, not observed previously in nature. In addition, HR-MAS NMR detected the N-linked glycan, GalNAc-α1,4-GalNAc-α1,4-[Glc-β1,3-]GalNAc-α1,4-GalNAc-α 1,4-GalNAc-α1,3-Bac, where Bac is 2,4-diacetamido-2,4,6-trideoxy-d-glucopyranose, in C. jejuni and Campylobacter coli. The presence of this common heptasaccharide in multiple campylobacter isolates demonstrates the conservation of the N-linked protein glycosylation pathway in this organism

Lipooligosaccharide, capsular polysaccharide, Campylobacter, NMR spectroscopy, mass spectrometry, magic angle

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7
4,5	bDRibf	106.2	81.2	70.7	84.0	63.0
4,2	Ac	174.9	22.9
4,3	Subst	54.8
4	bDGalfN	106.4	62.5	79.6	82.2	78.3	61.9
3,6	Me	60.4
3	aXDLglcHepp	98.1	72.2	73.8	70.2	72.3	79.5	63.1
6	x?GroN	61.3	53.9	61.3
	aDGlcpA	99.0	73.1	73.8	76.4	72.7	171.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7
4,5	bDRibf	5.35	4.21	4.30	4.13	3.70-3.89
4,2	Ac	-	2.05
4,3	Subst	3.75
4	bDGalfN	5.13	4.27	4.88	4.48	3.97	3.77-3.82
3,6	Me	3.55
3	aXDLglcHepp	5.61	3.53	3.72	3.56	4.08	3.80	3.86
6	x?GroN	3.67-3.72	4.03	3.67-3.72
	aDGlcpA	5.14	3.93	4.10	3.93	4.38	-


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7
4,5	bDRibf	106.2/5.35	81.2/4.21	70.7/4.30	84.0/4.13	63.0/3.70-3.89
4,2	Ac		22.9/2.05
4,3	Subst	54.8/3.75
4	bDGalfN	106.4/5.13	62.5/4.27	79.6/4.88	82.2/4.48	78.3/3.97	61.9/3.77-3.82
3,6	Me	60.4/3.55
3	aXDLglcHepp	98.1/5.61	72.2/3.53	73.8/3.72	70.2/3.56	72.3/4.08	79.5/3.80	63.1/3.86
6	x?GroN	61.3/3.67-3.72	53.9/4.03	61.3/3.67-3.72
	aDGlcpA	99.0/5.14	73.1/3.93	73.8/4.10	76.4/3.93	72.7/4.38	

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](O[C@H](CO)[C@@H]2O[C@@H](O[C@@H]3[C@@H](C(=O)NC(CO)CO)O[C@H](*)[C@H](O)[C@H]3O[C@@H]3O[C@@H]([C@@H](CO)OC)[C@H](O)[C@@H](O)[C@@H]3O)[C@H](NC(C)=O)[C@H]2OP(N)(=O)OC)O[C@H](CO)[C@H]1O

883.744 g/mol (C₃₁H₅₄R₂N₃O₂₄P, R = next and previous repeats, not counted in mol weight)