METACyt Biochemical Analysis Center, Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
J Proteome Res. 2010 Jul 2;9(7):3598-607. doi: 10.1021/pr100129n.
The assignment of protein glycosylation sites and their microheterogeneities are of biological importance, yet such characterization is still considered to be analytically very challenging. Several approaches have been recently developed to improve the characterization of glycosylation sites of proteins, including lectin and HILIC enrichment-based methods coupled to mass spectrometry. However, unequivocal assignment of protein glycosylation remains to be a daunting task, prompting continuous demands for the development of sensitive and cutting-edge analytical approaches. beta-N-Acetylglucosaminidase (endo-beta-GlcNAc-ases, Endo-M) is an endoglycosidase capable of hydrolyzing N,N'-diacetylchitobiose moiety in N-linked oligosaccharides bound to the asparagine amino acid residue in various glycoproteins. An attractive feature of this enzyme is its ability to cleave the N,N'-diacetylchitobiose moiety while leaving an N-acetylglucosamine residue bound to the protein. This enzyme is also known to be inactive in the presence of core fucose residue linked to the reducing-end N-acetylglucosamine residue (GlcNAc). Here, we describe an approach capitalizing on these features of Endo-M to (a) determine the glycosylation sites of proteins and the occupancy of these sites, and (b) determine the attachment sites of fucose residue containing N-glycans. The latter is important because of its biological implications. Tryptically digested glycoproteins, which were subjected to Endo-M treatment, were analyzed by LC-MS/MS. Systematic evaluation of the activity of Endo-M toward different glycan structures indicated a dependence of enzyme activity on the complexity of the glycan structures. Efficient release of N-glycans using Endo-M is only achieved through the inclusion of a battery of exoglycosidases to reduce the complexity of the attached glycans and subsequently prompt an effective enzymatic release. Upon Endo-M/exoglycosidase treatment of tryptically digested glycoproteins, glycosylated sites retain GlcNAc residue. The resulting peptides with GlcNAc residues attached to the glycosylation sites are easily assigned through LC-MS/MS analysis and subsequent database searching of the generated tandem MS of such entities. Comparing the LC-MS/MS results of the tryptic digest of glycoproteins treated with PNGase F and Endo-M/exoglycosidases allowed the assignment of core fucose residues to N-glycan reducing-ends. The detection of glycosylation sites only in the tryptic digest of PNGase F treated samples suggested core fucosylation of the attached N-glycans to such sites. This strategy was initially validated using model glycoproteins. It also proved to be useful in determining the glycosylation sites of blood serum glycoproteins.
蛋白质糖基化位点及其微观不均一性具有重要的生物学意义，但此类特征分析仍被认为极具挑战性。最近开发了几种方法来改善蛋白质糖基化位点的特征描述，包括基于凝集素和 HILIC 富集的方法与质谱联用。然而，蛋白质糖基化的明确分配仍然是一项艰巨的任务，这促使人们不断要求开发灵敏和先进的分析方法。β-N-乙酰氨基葡萄糖苷酶（endo-β-GlcNAc-ases，Endo-M）是一种内切糖苷酶，能够水解结合在各种糖蛋白中天冬酰胺氨基酸残基上的 N-连接寡糖中的 N,N'-二乙酰壳二糖部分。该酶的一个吸引人的特点是它能够在 N-乙酰葡萄糖胺残基与蛋白质结合的情况下切割 N,N'-二乙酰壳二糖部分。该酶在存在与还原端 N-乙酰葡萄糖胺残基（GlcNAc）相连的核心岩藻糖残基时也已知是无活性的。在这里，我们描述了一种利用 Endo-M 的这些特性的方法：(a) 确定蛋白质的糖基化位点及其占据情况，以及 (b) 确定含有岩藻糖残基的 N-聚糖的附着位点。这一点很重要，因为它具有生物学意义。用 Endo-M 处理的胰蛋白酶消化的糖蛋白通过 LC-MS/MS 进行分析。对 Endo-M 对不同聚糖结构的活性进行系统评估表明，酶活性取决于聚糖结构的复杂性。只有通过包含一系列外切糖苷酶来降低附着聚糖的复杂性，并随后促使有效的酶释放，才能有效地释放 N-聚糖。在胰蛋白酶消化的糖蛋白经 Endo-M/外切糖苷酶处理后，糖基化位点保留 GlcNAc 残基。通过 LC-MS/MS 分析和随后对这些实体的生成串联质谱的数据库搜索，很容易确定附着在糖基化位点上的 GlcNAc 残基的肽。比较用 PNGase F 和 Endo-M/外切糖苷酶处理的糖蛋白胰蛋白酶消化物的 LC-MS/MS 结果，可以将核心岩藻糖残基分配到 N-聚糖的还原端。只有在用 PNGase F 处理的样品的胰蛋白酶消化物中检测到糖基化位点，这表明附着的 N-聚糖在这些位点发生了核心岩藻糖基化。该策略最初使用模型糖蛋白进行了验证。它还被证明可用于确定血清糖蛋白的糖基化位点。
