PNGase F N-glycan Releasing Kit

Titration of PNGase F on RNase B (Rb) and Cy5-Labeled RNase B (Cy5-Rb) using bothnative and denaturing assay protocols. Deglycosylation of Rb (A) and Cy5-Rb (B) with PNGase F N-glycan Releasing Kit(Catalog # EA006) using both the native and denaturing assay protocols. >95%deglycosylation was achieved with 1000 ng and 1.6 ng of PNGase F under native and denaturingconditions, respectively. Digested samples were separated on 17% SDS gel. A. Rb (1 μg perdigestion) was digested with indicated amounts of PNGase F and imaged with silver staining.B. Cy5-Rb (0.2 μg for digestion) was digested with indicated amounts of PNGase F and visualized with fluorescent imaging. Labeling on RNase B was introduced by serial treatment with Recombinant HumanN-Acetylglucosaminyltransferase1/MGAT1 (8334-GT), UDP-GlcNAc,Recombinant Human B4GalT1 Protein (3609-GT), UDP-Gal, Recombinant HumanST6GAL1 (aa44-406) Protein (7620-GT), CMP-Cy5-Sialic Acid (ES302)(Wu, Z. et al. (2020) Glycobiology 30:970). Denaturing increased the PNGase F sensitivity by 500-fold.

Titration of PNGase F on Cy5-Neu5Ac Labeled RBD Protein (Cy5-RBD) using both native and denaturing assay protocols. Deglycosylation of Cy5-RBD with PNGase F N-glycan Releasing Kit (Catalog # EA006) using both the native and denaturing assay protocols. Cy5-RBD (1 µg per digestion) was treated with indicated amounts of PNGase F in 20 µL under both denaturing and native conditions at 37 °C for 60 minutes then separated on 17% SDS gel and imaged by trichloroethanol (TCE) staining (top panel, only proteins were visible, and the lower portion wascropped out) and fluorescent imaging (lower panel). >95% deglycosylation was achieved with 200 ng and 1.6 ng of PNGase F under native and denaturing conditions, respectively.

Titration of PNGase F on Cy5-Neu5Ac Labeled ACE-2 (Cy5-ACE-2) using both native and denaturing assay protocols. Deglycosylation of Cy5-ACE-2 with PNGase F N-glycan Releasing Kit (Catalog # EA006) using both the native and denaturing assay protocols Cy5-ACE-2. (1 µg for each digestion) was treated with indicated amounts of PNGase F in 20 µL under both denaturing and native conditions at 37 °C for 30 minutes then separated on 17% SDS gel and imaged by TCE staining (top panel, only proteins were visible, and the lower portion was cropped out) and fluorescent imaging (lower panel). Multiple bands are visible for the released glycans suggesting the complicated nature of the glycosylation on ACE-2. >90% deglycosylation was achieved with 2000 ng and 0.2 ng of PNGase F under native and denaturing conditions, respectively.

PNGase F pH profile test on Cy5-Neu5Ac Labeled RBD Protein (Cy5-RBD) using denaturing assay protocols. Denatured Cy5-RBD (1 µg per digestion) was treated with 5 ng of PNGase F at indicated pH in 20 µL at 37 °C for 60 minutes then separated on 17% SDS gel and imaged by TCE staining (top panel, only proteins were visible, and the lower portion was cropped out) and fluorescent imaging (lower panel). PNGase F showed activity from pH 5.0 to 10 with complete glycan removal between pH 6.0 to pH 8.5.

Mass spectrometry analysis of PNGase F treated bovine fetal RNase B. Native RNase B of different glycoforms with high-mannose glycans, Man-5 to Man-9, are indicated as Rb-Man5 to Rb-Man9 respectively. Upon PNGase F treatment under native condition, majority of these glycans were removed and resulted in deglycosylated RNase B (Rb). Mass spectrometry was performed using a Thermo Scientific Q Exactive HF system connected to a Thermo Vanquish LC. Samples were separated on a C4 column then analyzed using full scan at 240,000 resolution in intact mode. Data was analyzed using Thermo BioPharma Finder software.