Comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method
Section snippets
Fluorescent oligosaccharides
All derivatization reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA) except AA-Ac, which was synthesized from proflavin [20].
The desialylated, diantennary N-glycan A4A4 was obtained from bovine fibrin and purified by carbon HPLC as described previously [62]. The glycan pool probably contained a few percentage points of isomers with 3-linked galactose. The model oligosaccharide A4A4 was labeled with the various reagents according to published procedures as compiled in Table 1. After
Preparation of labeled N-glycans
A sufficiently large stock of the neutral, diantennary N-glycan A4A4 (with small amounts of isomers with 3-linked Gal) was prepared. Aliquots of 10 nmol were derivatized with various aromatic amines. In addition to the well-tried reagents PA and AB, several other labels described in the literature were used (see introductory paragraphs). Aminosalicyl acids were also tested in the hope of constituting a hydrophilic label (similar to PA) suitable for RP–HPLC with negative mode ESI–MS. Of the three
Discussion
In this broad comparison of glycan labeling reagents in a variety of applications, it is tempting to elect winners of single disciplines and finally an overall winner. The original and most important purpose of labeling is to render oligosaccharides detectable. Thus, it is at first surprising that, after two decades of reductive amination for glycan analysis, the most sensitive labels (Pro, ProA, and ABBE) have not yet found their use for this purpose. However, we found in this study that the
Acknowledgments
D.K. received a fellowship of the European Commission for the project “GMO-CARE.” We thank Martin Schott for technical assistance and Elise Langdon-Neuner for expert language editing.
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Current address: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia.