Rapid release of N-linked glycans from glycoproteins by pressure-cycling technology.
The standard, well-established sample preparation protocol to release N-linked glycans from glycoproteins for downstream analysis requires relatively long deglycosylation times (from several hours to overnight) and relatively high endoglycosidase concentration (from 1:250 to 1:500 enzyme:substrate molar ratio). In this paper, we significantly improve this standard protocol by the use of pressure-cycling technology (PCT) to increase the speed and decrease the relative amount of PNGase F during the release of N-linked glycans from denatured glycoproteins. With the application of pressure cycling from atmospheric to as high as 30 kpsi, >95% release of the asparagine-linked glycans from bovine ribonuclease B, human transferrin, and polyclonal human immunoglobulin was rapidly achieved in a few minutes using as low as 1:2500 enzyme:substrate molar ratio. The deglycosylation rate was first examined by SDS-PAGE at the protein level. The released glycans were then quantitated by capillary electrophoresis with laser induced fluorescence detection (CE-LIF). This new sample preparation protocol readily supports large-scale glycan analysis of biopharmaceuticals with rapid deglycosylation times.
Szabo
Zoltan
Z
Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.
Guttman
András
A
Karger
Barry L
BL
D000818:Animals; D002417:Cattle; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011312:Pressure
D006023:Glycoproteins; D007074:Immunoglobulin G; D011134:Polysaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase