PNGase F catalyzes de-N-glycosylation in a domestic microwave

Abstract

Common de-N-glycosylation protocols usually require a lengthy incubation time. Although pressure cycling technology or scientific microwave reactors can accelerate this enzyme reaction, they may not be easily accessible. In this brief report, we employed an alternative strategy using a standard domestic microwave oven to perform the de-N-glycosylation. Model glycoproteins (bovine RNase B, bovine fetuin, and human IgG) and a complex mixture from human plasma were fully deglycosylated in 20 min, without any apparent adverse affects on the glycans or protein backbones. This new method provides a simple and inexpensive solution to achieve rapid de-N-glycosylation.

Figure 1

SDS-PAGE of bovine RNase B (A); bovine fetuin (B); human IgG (C); and MARS-7 bound fractions from two human plasma samples (D & E); before and after PNGase F digestion by domestic microwave irradiation (DMW) or standard method (* indicates overnight incubation in a 37 °C oven). (A) to (D) were stained by coomasssie blue stains, and (E) was stained by Pro-Q glycoprotein stain. The band of PNGase F (~ 36K Da) was visible in figures (A to D).

Figure 2

MALDI-MS spectra of permethylated N-glycans from bovine RNase B (A); bovine fetuin (B); and human IgG (C) after the domestic microwave release protocol. Each peak was assigned a representative composition cartoon based on their m/z value and previous reports.