An analytical system for the characterization of highly heterogeneous mixtures of N-linked oligosaccharides

Abstract

A novel system for characterizing complex N-linked oligosaccharide mixtures that uses a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS), capillary electrophoresis (CE), and high-performance liquid chromatography (HPLC) has been developed. In this study, oligosaccharides released from recombinant TNK–tPA (tissue plasminogen activator) were derivatized with 5-amino-2-naphthalenesulfonic acid (ANSA). The negative charge imparted by the ANSA label facilitated the analysis of the oligosaccharides by MALDI–TOF MS by allowing the observation of both neutral and sialylated oligosaccharides in a single negative ion mode spectrum. Labeling with ANSA was also determined to be advantageous in the characterization of oligosaccharides by both HPLC and CE. The ANSA label was demonstrated to provide superior resolution over the commonly used label 8-aminopyrene-1,3,6-trisulfonic acid (APTS) in both the CE and HPLC analysis of oligosaccharides. To date, no other labels that enable the analysis of complex oligosaccharide mixtures in a single mass spectral mode, while also enabling high-resolution chromatographic and electrophoretic separation of the oligosaccharides, have been reported. By integrating the structural information obtained by MALDI–TOF MS analysis with the ability of CE and HPLC to discriminate between structural isomers, the complete characterization of complex oligosaccharide mixtures is possible.