Ultrasensitive profiling and sequencing of N-linked oligosaccharides usingstandard DNA-sequencing equipment

The analysis of protein-linked glycans is of increasing importance, both in basic glycobiological research and during the production process of glycop rotein pharmaceuticals. In many cases, the amount of glycoprotein available for typing the glycans is very low. This, combined with the high branching complexity typical for this class of compounds, makes glycan typing a chal lenging task. We present here methodology allowing the medium-throughput an alysis of N-glycans derived from low picomole amounts of glycoproteins usin g the standard DNA-sequencing equipment available in any life sciences labo ratory, The high sensitivity of the overall analytical process (from glycop rotein to results) is obtained using state-of-the-art deglycosylation proce dures combined with a highly efficient and reproducible novel postderivatiz ation cleanup step involving Sephadex G10 packed 96-well filterplates, All sample preparation steps (enzymatic deglycosylation with PNGase F, desaltin g, derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid, and postderiva tization cleanup) are performed using 96-well-based plates. This integrated sample preparation scheme is also compatible with capillary electrophoresi s and MALDI-TOF-MS platforms already in use in some glycobiology labs and a nticipates the higher throughput that will be offered by the capillary-arra y-based DNA sequencers currently penetrating the market. The described tech nology should bring high-performance glycosylation analysis within reach of each life sciences lab and thus help expedite the pace of discovery in the field of glycobiology.