Glycoproteins are biologically active proteins of which the attached glycans contribute to their biological functionality. Limited data is available on the functional properties of these N-glycans in isolation, without the protein core. Glycan release, typically performed with the PNGase F enzyme, is achieved on denatured proteins in the presence of detergents which are notoriously difficult to be completely removed. In this work we compared two methods aiming at recovering N-glycans in a high yield and at high purity from a PNGase F glycoprotein digest of bovine lactoferrin. Detergents were removed from the digest by two separate approaches. In the first approach, protein and glycans were precipitated with acetone and the detergent containing supernatant was discarded. In the second approach, detergent was removed by adsorption onto a polystyrene resin. Following detergent removal, the glycans were further purified by a sequence of solid phase extraction (SPE) steps. Both approaches for detergent removal yielded a final glycan purity above 85%. Recovery of the glycans from lactoferrin was, however, much lower when utilizing acetone precipitation versus the polystyrene resin; 52% versus 85% respectively. A more detailed analysis of the acetone precipitation step revealed a loss of shorter oligomannose structures specifically. A loss of glycans of lesser complexity (oligomannose and biantennary structures) was also observed for other glycoproteins (RNase B, porcine thyroglobulin, human lactoferrin). These results indicate that acetone precipitation, a commonly used step for small-scale glycan purification, is not suitable for all target glycoproteins. The polystyrene resin detergent removal step conserved the full N-glycan profile and could be applied to all mammalian glycoproteins tested. Using this optimized protocol, large-scale quantitative isolation of N-glycan structures was achieved with sufficient purity for functional studies.
Keywords: Acetone precipitation; Detergent removal; Glycan purification; Lactoferrin; N-Linked protein glycosylation.
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