Electrospray ionization-ion trap mass spectrometry, with its capacity to perform multiple stages of fragmentation (MSn), is demonstrated as an effective method for the structural characterization of permethylated N-linked complex glycoprotein oligosaccharides. Complex glycan structural features, such as N-acetyllactosamine antenane, neuraminic acids, and nonreducing terminal GlcNAc monosaccharides, commonly suppress cross-ring and core saccharide cleavages in traditional MS/MS experiments. Using ion trap mass spectrometry, removal of these substituents permits determination of branching patterns and intersaccharide linkages by MS3 and MS4. Both sequence and linkage data are obtained for N-acetyllactosamine and sialyl-N-acetyllactosamine oligosaccharide antennae from biantennary glycans using MS3, and the location of a bisecting GlcNAc residue is also established after exposing the core pentasaccharide. Higher-order experiments further illustrate the potential of electrospray ionization-quadrupole ion trap mass spectrometry for carbohydrate analysis, as MS8 is used to produce significant and otherwise unobtainable branching information for an oligosaccharide from chicken ovalbumin. These studies constitute further evidence of the unique role that ion trap mass spectrometry can assume in the area of oligosaccharide analysis.

The use of electrospray ionization-quadrupole ion trap mass spectrometry for the characterization of linear oligosaccharides and N-linked protein oligosaccharide mixtures is described. Tandem mass spectrometry (MS/MS) experiments with orders higher than two offer a number of ways to enhance MS/MS spectra and to derive information not present in MS and MS2 spectra. Three such methods are presented in this paper. (a) Collisional activation of permethylated oligosaccharide molecular ions (MS2) as illustrated by maltoheptaose, produces abundant fragments from glycosidic bond cleavages which indicate composition and sequence, and weak cross-ring cleavage products which denote specific linkages within the oligosaccharide. Through the trapping and further dissociation of these fragments (MSn), cross-ring cleavage products can be confirmed and their relative abundances increased to facilitate interpretation. (b) The mechanisms of formation of two isobaric ions or ions isobaric with another ion's isotope peaks, such as those present in the MS2 spectrum of the ribonuclease B oligosaccharide GlcNAc2-Man5 can be independently established by separate MS3 experiments. (c) Ions in the MS2 spectrum, specific for individual components of an isobaric mixture, can be isolated and characterized by further stages of fragmentation. This is illustrated by two isobaric oligosaccharides from chicken ovalbumin of the composition HexNAc5Hex5. These findings indicate the utility of ion trap mass spectrometry towards the facile determination of oligosaccharide composition, sequence, branching and linkage, providing a wealth of structural information not obtainable by other individual methods of carbohydrate mass spectrometric analysis.

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We recently identified 1K K,25-dihydroxy-3-epi-vitamin D Q as a major in vitro metabolite of 1K K,25-dihydroxyvitamin D Q , produced in primary cultures of neonatal human keratinocytes. We now report the isolation of 1K K,25-dihydroxy-3-epi-vitamin D Q from the serum of rats treated with pharmacological doses of 1K K,25-dihydroxyvitamin D Q . 1K K,25-dihydroxy-3-epi-vitamin D Q was identified through its co-migration with synthetic 1K K,25-dihydroxy-3-epi-vitamin D Q on both straight and reverse phase high performance liquid chromatography systems and by mass spectrometry. Along with 1K K,25-dihydroxy-3-epi-vitamin D Q , other previously known metabolites, namely, 1K K,24(R),25-trihydroxyvitamin D Q , 1K K,25-dihydroxy-24-oxo-vitamin D Q and 1K K,25-dihydroxyvitamin D Q -26,23-lactone, were also identified. Thus, our study for the first time provides direct evidence to indicate that 1K K,25-dihydroxy-3-epi-vitamin D Q is an in vivo metabolite of 1K K,25-dihydroxyvitamin D Q in rats.z 1999 Federation of European Biochemical Societies.