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

New England Biolabs（NEB）公司       NEB公司——三十多年的卓越品质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。重组酶NEB公司对酶的生产与其基础科研不能分开，通过对限制/修饰系统的克隆和过量表达方面的研究，使我们能够大大降低成本，改善产品质量。NEB已经成功克隆了180多种内切酶，其中大多是完全克隆，少数是部分克隆。目前，NEB可供应240多种内切酶，其中180多种可以重组酶形式提供，同时还有大量的应用途广泛的重组聚合酶和重组修饰酶。质量与客户服务NEB公司凭借着严格的质检程序、深入的基础科研以及不断开发的研发项目，承诺为全球科技人员提供高纯度的科研产品。直接与NEB总部联系或与NEB的国际网点联系，即可体验到NEB的个性化客户服务。公司内负责生产以及负责进行质量监控的科学家（产品负责人）也就是技术支持人员。他们为客户解答有关限制核酸内切酶、甲基化酶、以及其他DNA修饰酶、蛋白质修饰酶方面的问题。同时，公司的有机合成部门可提供linkers、引物、adaptors、探针以及寡核苷酸合成等方面的信息。研究人员也可为客户提供技术支持服务，这些研究人员在DNA测序、甲基化、克隆、过量表达、发酵、蛋白质纯化以及蛋白质分析方面都有很深造诣。基础科研NEB在分子生物学和寄生虫学方面的基础研究由公司内部的资深科学家负责。这些科学家在他们各自的领域都卓有成绩，经常在业内著名杂志上发表文献，指导博士后工作、为大学生提供暑期实习机会，而且经常受邀去当地学校进行演讲。NEB鼓励公司与外界合作，每周学术探讨会为其他科学家们提供了交流和展示的平台。环保政策NEB公司在资助科研项目时最基本原则之一就是：一切工作都应符合保护生态环境。公司产品生产、分析以及运输过程既要满足产品的稳定性，也要符合我们的环保政策。20年来，NEB公司最引人注目的一项环保政策是：回收运输泡沫盒，虽然这项工作首创于美国，但是，目前加拿大、德国以及英国的分公司也同样实施了。公司还在内部开展多方位的回收工作，经常用再生纸张印刷市场宣传资料。这些小细节不会影响产品质量，但是对环境的保护却是显而易见。公司职责除环保政策外，通过基金会，NEB还竭力为改善当地社区及全人类尽微薄之力。NEB基金会创立于1982年，属私人性质，其宗旨是支持发展中国家的环保、教育、健康及艺术事业。此外，NEB捐款委员会也经常向当地社区进行捐款活动。