Department of Nutrition, Harvard University School of Public Health, Boston, Massachusetts 02115, USA.
In the preceding paper, preliminary analysis revealed a new type of O-linked oligosaccharide of 1244 Da at each of two proposed glycosylation sites on several proteins secreted by the Gram-negative bacterium Flavobacterium meningosepticum (Plummer, T. H., Jr., Tarentino, A. L., and Hauer, C. R. (1995) J. Biol. Chem. 270, 13192-13196). In this report we detail the linkage, sequence, and branching of this unusual heptasaccharide by electrospray (ES) ionization mass spectrometry (MS), and collision-induced dissociation (CID). The proposed structure was supported by a combination of isotopic labeling, composition and methylation analysis, and the preparation of several chemical analogs and derivatives with each product evaluated by MS and CID. The singly branched structure contained seven residues, including three different uronyl analogs: a methylated rhamnose and mannose, a glucose, and a reducing terminal mannose. Only pyranose ring forms were detected ((2-OMe)Man1-4GlcNAcU1-4GlcU1-4Glc1-4(2-OMe)G lcU-4 [(2-OMe)Rham1-2]Man).