This communication reports the development of an LC/MS platform for the analysis of permethylated oligosaccharide alditols that, for the first time, demonstrates routine online oligosaccharide isomer separation of these compounds before introduction into the mass spectrometer. The method leverages a high-resolution liquid chromatography system with the superior fragmentation pattern characteristics of permethylated oligosaccharide alditols that are dissociated under low-energy collision conditions using quadrupole orthogonal time-offlight (QoTOF) instrumentation and up to pseudo MS 3 mass spectrometry. Glycoforms, including isomers, are readily identified and their structures assigned. The isomer-specific spectra include highly informative cross-ring and elimination fragments, branch position specific signatures, and glycosidic bond fragments, thus facilitating linkage, branch, and sequence assignment. The method is sensitive and can be applied using as little as 40 fmol of derivatized oligosaccharide. Because permethylation renders oligosaccharides nearly chemically equivalent in the mass spectrometer, the method is semiquantitative and, in this regard, is comparable to methods reported using high field NMR and capillary electrophoresis. In this postgenomic age, the importance of glycosylation in biological processes has become clear. The nature of many of the important questions in glycomics is such that sample material is often extremely limited, thus necessitating the development of highly sensitive methods for rigorous structural assignment of the oligosaccharides in complex mixtures. The glycomics platform presented here fulfills these criteria and should lead to more facile glycomics analyses. (J Am

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We describe a new Type II congenital disorder of glycosylation (CDG-II) caused by mutations in the conserved oligomeric Golgi (COG) complex gene, COG8. The patient has severe psychomotor retardation, seizures, failure to thrive and intolerance to wheat and dairy products. Analysis of serum transferrin and total serum N-glycans showed normal addition of one sialic acid, but severe deficiency in subsequent sialylation of mostly normal N-glycans. Patient fibroblasts were deficient in sialylation of both N- and O-glycans, and also showed slower brefeldin A (BFA)-induced disruption of the Golgi matrix, reminiscent of COG7-deficient cells. Patient fibroblasts completely lacked COG8 protein and had reduced levels and/or mislocalization of several other COG proteins. The patient had two COG8 mutations which severely truncated the protein and destabilized the COG complex. The first, IVS3 + 1G > A, altered the conserved splicing site of intron 3, and the second deleted two nucleotides (1687-1688 del TT) in exon 5, truncating the last 47 amino acids. Lentiviral-mediated complementation with normal COG8 corrected mislocalization of other COG proteins, normalized sialylation and restored normal BFA-induced Golgi disruption. We propose to call this new disorder CDG-IIh or CDG-II/COG8.