A fast and convenient solid phase preparation method for releasing N-glycans from glycoproteins using trypsin- and peptide-N-glycosidase F (PNGase F)-impregnated polyacrylamide gels fabricated in a pipette tip.
J Pharm Biomed Anal
; 179: 112995, 2020 Feb 05.
Article
em En
| MEDLINE
| ID: mdl-31767225
ABSTRACT
An efficient deglycosylation process is a key requirement for the identification and characterization of glycosylation during the production and purification of therapeutic antibodies. PNGase F is widely used for the deglycosylation of N-linked glycans. The commonly-used in-solution deglycosylation method is relatively time-consuming and requires several hours up to overnight for complete removal of all N-linked glycans. In order to develop a simple and efficient method for the rapid release of N-linked glycans from glycoproteins, we fabricated trypsin- and PNGase F-impregnated polyacrylamide gels in a commercial 200⯵L volume pipette tip. Our enzyme reactor is based on simple photochemical copolymerization of monomers using the following procedure (1) a pipette tip was filled with a gel solution comprising acrylamide, N,N'-methylene-bis-acrylamide containing PNGase F or trypsin with 2,2-azobis(2-methyl-N-(2-hydroxyethyl) propionamide) as a photocatalytic initiator; and (2) in situ polymerization of gel solution approximately 30â¯mm from the tip was performed by irradiation with a 365â¯nm blue LED beam from a distance 10â¯mm. The fixed enzymes maintained their activities in the polyacrylamide gel and the reaction was completed by 40 iterations of suction and discharge with a pipette (hereafter referred to as manual pipetting times) for 8â¯min with each enzyme digestion. Capillary electrophoresis (CE) of released glycans labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) demonstrated quantitative recovery of glycans from selected glycoproteins.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polissacarídeos
/
Resinas Acrílicas
/
Glicoproteínas
/
Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase
Tipo de estudo:
Guideline
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article