Determination of N-glycans in glycoproteins using chemoenzymatic labeling with Endo-M N175Q

A novel approach for the determination of N-glycans in glycoproteins has been developed based on a combination of deglycosylation, chemoenzymatic labeling, and high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Glycoproteins were digested with PNGase F and then the released N-glycans were enriched and separated from the digesting solution using ultrafiltration units according to the molecular mass differences. The N-glycans were analyzed by HPLC/ESI-MS after chemoenzymatic labeling with Endo-M N175Q an Endo-M mutant. We have shown that the Endo-M N175Q demonstrates a noticeable transglycosidase-like activity for natural N-glycans. The productivity of Endo-M N175Q is estimated to be 2.0-fold higher than that of Endo-M. The experimental strategy was validated using glycoproteins with known glycan structures such as ovalbumin and ribonuclease B. The proposed method offers a promising advance for determining N-glycans in glycoproteins.