Glycosylation is one of the most common post-translational modifications of proteins, as shown in Figure 1. N-linked glycosylation occurs when glycans are attached to asparagine residues on the core protein. O-linked glycosylation occurs when glycans are attached to serine or threonine residues. Both chemical and enzymatic methods exist for removing oligosaccharides from glycoproteins. However, chemical methods such as β-elimination with mild alkali or mild hydrazinolysis can be harsh and may result in incomplete sugar removal and degradation of the protein; whereas, enzymatic methods are much gentler and can provide complete sugar removal with no protein degradation.PNGase F is the most effective enzymatic method for removing almost all N-linked oligosaccharides from glycoproteins. PNGase F digestion deaminates the aspargine residue to aspartic acid, and leaves the oligosaccharide intact, keeping it suitable for further analysis. Oligosaccharides containing a fucose α(1-3)-linked to the glycan core are, however, resistant to PNGase F which can occur on some plant and insect glycoproteins. To remove O-linked glycans, monosaccharides must be removed by a series of exoglycosidases until only the Galβ1-3GalNAc (core 1) and/or the GlcNAcβ1-3GalNAc (core 3) cores remain attached to the serine or threonine. NEB’s O-Glycosidase, cloned from Enterococcus faecalis, can then remove these core structures with no modification of the serine or threonine residues. Any modification of the core structures, including sialyation, will block the action of the O-Glycosidase. Sialic acid residues are easily removed by a general α2-3,6,8,9 Neuraminidase A. In addition, exoglycosidases such as β(1-4)Galactosidase S and β-N-Acetylhexosaminidase_f can be included in deglycosylation reactions to remove other complex modifications often known to be present on the core structures. This combination of enzymes may not remove all O-linked oligosaccharides but should remove many common oligosaccharide structures.The Protein Deglycosylation Mix II contains all of the enzymes, reagents, and controls needed to remove all N-linked and simple O-linked glycans as well as some complex O-linked glycans. This mix contains enzyme sufficient for 20 reactions or the cleavage of as much as 2 mg of glycoprotein. All of the enzymes and reagents included in the Protein Deglycosyation Mix II are Mass Spectrometry compatible. Following the deglycosylation reaction, samples are ready to be prepared for mass spectrometry analysis.Figure 1:A Glycoprotein modified with O-linked and N-linked glycosylation.

Figure 2

Protein Deglycosylation Mix II:PNGase F (Glycerol-free), Recombinant:10,000 units/vialO-Glycosidase:80,000 units/vial α2-3,6,8,9 Neuraminidase A:400 units/vialβ1-4 Galactosidase S:960 units/vialβ-N-acetylhexosaminidase_f:300 units/vialSubstrate Control: Fetuin, 0.5 mg (Fetuin contains sialylated N-linked and O-linked glycans)Description of Enzymes Included in the Protein Deglycosylation Mix IIO-Glycosidase (NEB #P0733), also known as Endo-α-N-Acetylgalactosaminidase, is a recombinant enzyme cloned from Enterococcus faecalis (1). It catalyzes the removal of core 1 and core 3 O-linked disaccharides from glycoproteins. The molecular weight is approximately 147 kDa.PNGase F (Glycerol-free), Recombinant (NEB #P0709), also known as Peptide: N-glycosidase F, is cloned from Elizabethkingia miricola (formerly Flavobacterium meningosepticum) and expressed in E. coli (2). PNGase F (Glycerol-free), Recombinant is an amidase which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins unless α(1-3) core fucosylated. The molecular weight is approximately 36 kDa.α2-3,6,8,9 Neuraminidase A (NEB #P0722), also known as Sialidase A, is a recombinant enzyme cloned from Arthrobacter ureafaciens and expressed in E. coli (3). It catalyzes the hydrolysis of α2,3, α2,6, α2,8 and α2,9 linked N-acetylneuraminic acid residues from glycoproteins and oligosaccharides. The molecular weight is approximately 100 kDa.β1-4 Galactosidase S (NEB #P0745), is a recombinant enzyme cloned from Streptococcus pneumoniae and expressed in E. coli (4). It is a highly specific exoglycosidase that catalyzes the hydrolysis of β1-4 linked galactose residues from oligosaccharides. The molecular weight is approximately 231 kDa.β-N-Acetylhexosaminidase_f (NEB# P0721), is a recombinant enzyme cloned from Streptomyces plicatus (5) and overexpressed in E. coli (6). It catalyzes the hydrolysis of terminal β-N-acetylgalactosamine and glucosamine residues from oligosaccharides. The molecular weight is approximately 100 kDa.

This product is related to the following categories:

Exoglycosidases Products,

Endoglycosidases Products,

Proteome Analysis Products

This product can be used in the following applications:

Expression Systems,

Protein Digestion,

Glycan Sequencing,

Recombinant Glycoprotein Expression,

Glycoprotein Analysis

NEB公司成立于二十世纪七十年代中期，拥有众多经验丰富的科学家，是生产生命科学试剂的领导者。目前，NEB为基因组研究提供最齐全的重组酶和天然酶，并且公司业务范围已延伸至蛋白质组学和药品开发领域。回顾三十余年来的历程，NEB公司作为先驱公司之一，为促进生物科技工业的发展做出了巨大的贡献。NEB美国总部乔 迁新址后拥有最尖端的设备，有一座现代化的发酵中心及设备齐全的实验室，这些实验室主要用于产品生产、质量监控、 产品开发和基础科研之用。作为首批以商业规模生产限制性内切酶的公司之一，NEB一直专注于内切酶的研究，并保持 业内领先水平。NEB公司一贯坚持以科学为本的原则，公司生产的试剂因其高质量、高性价比享誉世界。NEB（北京）有限公司 New England Biolabs (Beijing) LTD. NEB（北京）有限公司New England Biolabs (Beijing) LTD. 为美国New England Biolabs, Inc.在华投资兴建的独资子公司，负责处理其在中国内地及香港地区的全部业务，同时承担部分新产品的研发工作。纽英伦生物技术（北京）有限公司成立于2001年11月，为中国生命科学研究者提供一流的产品和专业的技术服务，促进国内外生命科学学术交流，与中国科研工作者一起将中国推入到二十一世纪生命科学的前沿中去。