Assigning N-Glycosylation Sites of Glycoproteins Using LC/MSMS in Conjunction with Endo-M/Exoglycosidase Mixture
Assigning N-Glycosylation Sites of Glycoproteins Using LC/MSMS in Conjunction with Endo-M/Exoglycosidase Mixture
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DOI：
10.1021/pr100129n
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发表时间：
2010-07-01
影响因子：
4.4
通讯作者：
Mechref, Yehia
中科院分区：
文献类型：
--
作者：
Segu, Zaneer M.;Hussein, Ahmed;Mechref, Yehia
蛋白质糖基化位点及其微异质性的分配具有生物学重要性，但这种表征仍然被认为是分析上非常具有挑战性的。最近已经开发了几种方法来改善蛋白质糖基化位点的表征，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，促使人们不断要求开发灵敏和尖端的分析方法。β-N-乙酰葡糖胺糖苷酶（内切-β-GlcNAc-酶，Endo-M）是一种内切糖苷酶，能够水解与各种糖蛋白中的天冬酰胺氨基酸残基结合的N-连接寡糖中的N，N ′-二乙酰壳二糖部分。这种酶的一个吸引人的特征是它能够切割N，N '-二乙酰壳二糖部分，同时留下与蛋白质结合的N-乙酰葡糖胺残基。还已知该酶在与还原末端N-乙酰葡糖胺残基（GlcNAc）连接的核心岩藻糖残基存在下无活性。在这里，我们描述了一种利用Endo-M的这些特征的方法，以（a）确定蛋白质的糖基化位点和这些位点的占有率，以及（B）确定含有N-聚糖的岩藻糖残基的附着位点。后者很重要，因为它具有生物学意义。胰蛋白酶消化的糖蛋白，这是进行Endo-M处理，通过LC-MS/MS进行了分析。Endo-M对不同的聚糖结构的活性的系统评价表明酶活性的聚糖结构的复杂性的依赖性。使用Endo-M的N-聚糖的有效释放仅通过包含一系列外切聚糖酶来实现，以降低所连接的聚糖的复杂性并随后促进有效的酶促释放。胰蛋白酶消化的糖蛋白经内切-M/外切糖苷酶处理后，糖基化位点保留GlcNAc残基。通过LC-MS/MS分析和随后对此类实体生成的串联MS进行数据库搜索，可以轻松地对所得到的具有连接至糖基化位点的GlcNAc残基的肽进行归属。比较用PNGase F和内切-M/外切糖苷酶处理的糖蛋白的胰蛋白酶消化物的LC-MS/MS结果，可以将核心岩藻糖残基指定为N-聚糖还原末端。仅在PNGase F处理样品的胰蛋白酶消化物中检测到糖基化位点，表明连接至这些位点的N-聚糖的核心岩藻糖基化。该策略最初使用模型糖蛋白进行验证。它也被证明是有用的，在确定血清糖蛋白的糖基化位点。
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.