Mass spectrometric studies on glycoprotein oligosaccharides: A modified procedure for the liquid secondary ion mass spectrometric analysis of glycoprotein oligosaccharides. Studies on the nature of glycosylation on baculovirus-expressed mouse interleukin-

Reductive amination with n-hexylamine was used as a derivatization procedure to improve the mass spectral sensitivity of oligosaccharides during liquid secondary ion mass spectrometry (LSIMS). Experiments with maltoheptaose showed that an improvement of 40 fold in detectability, during positive ion LSIMS, can be obtained over that of the native oligosaccharide using this derivative. The molecular ion can be observed with 13 pmoles of derivatized oligosaccharide with a signal to background ratio of 2/1. Permethylation of the n-hexylamine-derivatized maltoheptaose affords an oligosaccharide in which exhaustive methylation at the newly formed secondary nitrogen forms a quaternary ammonium salt. When this is subjected to positive ion LSIMS, an abundant M{dollar}sp+{dollar} ion is observed with excellent mass spectral sensitivity. A signal to background ratio of 78/1 was observed when 5 pmoles of the oligosaccharide was used for the analysis. This procedure, reductive amination with n-hexylamine followed by permethylation, was applied to the oligosaccharides on model glycoproteins, human transferrin and bovine ribonuclease B. The procedure was then used for the positive ion LSIMS analysis of the oligosaccharides on a baculovirus-expressed (BmNPV) mouse interleukin-3 produced in Bombyx mori.; An integrated approach of mass spectrometry, high pH anion exchange chromatography, and enzymatic analysis was used for the study of the oligosaccharides on BmNPV-expressed mouse interleukin-3. This recombinant lymphokine was found to be glycosylated with a single major Asn-linked oligosaccharide with the truncated structure of Man{dollar}alpha{dollar}(1-6)Man{dollar}beta{dollar}(1-4)GlcNAc(1-4)(Fuc{dollar}alpha{dollar}(1-6)) GlcNAc. A minimum of five other truncated core oligosaccharides containing two to four mannose residues were observed.