Methods for the absolute quantification of N-glycan biomarkers.
Glycan arrays have become the premier tool for rapidly establishing the binding or substrate specificities of lectins and carbohydrate-processing enzymes. New approaches for accelerating carbohydrate synthesis to address the enormous complexity of natural glycan structures are necessary. Moreover, optimising glycan immobilisation is key for the development of selective, sensitive and reproducible array-based assays. We present a tag-based approach that accelerates the preparation of glycan arrays on all levels by improving the synthesis, the purification and immobilisation of oligosaccharides. Glycan primers were chemically attached to bifunctional polyethyleneglycol (PEG) tags, extended enzymatically with the help of recombinant glycosyltransferases and finally purified by ultrafiltration. When printed directly onto activated glass slides, these glycoPEG tags afforded arrays with exceptionally high sensitivity, low background and excellent spot morphology. Likewise, the conjugation of glycoPEG tags to latex nanoparticles yielded multivalent scaffolds for carbohydrate-binding assays with very low non-specific binding.