Rapid release of N-linked glycans from glycoproteins by pressure-cycling technology

Abstract

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.

Figure 1

The effect of the maximum pressure level of pressure cycling on PNGase F mediated deglycosylation of the N-linked sugars from RNase B (Coomassie Brilliant Blue stained SDS-PAGE image). The bands at ~15 kDa and ~18 kDa represent the deglycosylated and intact forms of RNase B, respectively. Deglycosylation of denatured RNase B was carried out at 37°C for 5 minutes with 1:2500 enzyme:substrate molar ratio in the presence of Triton X-100. Control: 5 min deglycosylation at atmospheric pressure and 37°C. Pressure cycles: 50 s pressure/10 s atmospheric. Left lane: SDS-PAGE protein sizing standards: aprotinin (6 kDa), lysozyme 14 kDa), myoglobin (17 kDa) and carbonic anhydrase (28 kDa).

Figure 2

Comparative CE analysis of APTS labeled released glycans from RNase B and human transferrin by PCT and atmospheric deglycosylation using 1:2500 enzyme:substrate molar ratio. A) APTS labeled maltooligosaccharide ladder; B) RNase B glycans released at 30 kPsi pressure cycling level for 5 min (peaks in the order of their increasing migration times: Man 5 – Man 9); C) RNase B glycans released at atmospheric pressure for 3 hours; D) RNase B glycans released at atmospheric pressure for 5 minutes; E) transferrin glycans released at 30 kPsi pressure cycling level for 20 minutes; F) transferrin glycans released at atmospheric pressure for 3 hours; G) transferrin glycans released at atmospheric pressure for 20 minutes; H) asialotransferrin glycans released at atmospheric pressure for 3 hours. IS1: APTS derivatized maltopentaose, IS2: APTS derivatized maltose. Man 5, A2, A2F, A1 and NA2 structures are shown in Scheme 1. All separations were carried out in a PVA coated capillary (effective length: 40 cm, i.d.: 50 μm) at ambient temperature in 25 mM acetate (pH 4.75) buffer applying 250 V/cm field strength. Injection: hydrostatic, Δ 10 cm/10 sec.

Figure 3

Comparative CE analysis of APTS labeled released glycans from polyclonal human IgG using PCT and atmospheric deglycosylation with 1:2500 enzyme:substrate molar ratio. Traces: A) APTS labeled maltooligosaccharide ladder; B) IgG glycans released at 30 kPsi pressure cycling level for 5 min; C) IgG glycans released at atmospheric pressure for 3 hours; D) IgG glycans released at atmospheric pressure for 5 minutes. IS2: APTS derivatized maltose. All other conditions were the same as in Fig 2. Structures of G0, G1, G1′ and G2 are shown in Scheme 1.

Scheme 1

Schematic representation of the glycan structures used. Symbols adapted from, ³⁴ linkages from right to left.