An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins
An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins
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DOI：
10.1021/pr0700605
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发表时间：
2007-08-01
影响因子：
4.4
通讯作者：
Bunkenborg, Jakob
中科院分区：
文献类型：
--
作者：
Hagglund, Per;Matthiesen, Rune;Bunkenborg, Jakob
蛋白质糖基化的全局蛋白质组分析是一个重大挑战，由于这种翻译后修饰的固有的异质性和多样性。因此，通常在质谱分析之前酶促去除连接到蛋白质或肽链的聚糖，从而降低复杂性并促进糖基化位点测定。在这里，我们使用了两种不同的酶促去糖基化策略进行N-糖基化位点分析。(1)通过肽-N-糖苷酶（PNGase）消化去除整个N-聚糖链，同时对释放的天冬酰胺残基进行脱酰胺。该反应在（H2O）-O-18中进行，以便于通过O-18取代形成的天冬氨酸残基来鉴定先前的糖基化肽。(2)用两种内切-β-N-乙酰葡糖胺糖苷酶（Endo D和Endo H）消化，该酶切割保守N-聚糖核心结构中两个N，乙酰葡糖胺（GlcNAc）残基之间的糖苷键，留下单个GlcNAc残基，推定岩藻糖基侧链与肽连接。为了能够消化复杂和杂合型N-聚糖，还包括许多外切糖苷酶（β-半乳糖苷酶、神经氨酸酶和N-乙酰基-β-氨基葡糖苷酶）。这两种策略在此应用于鉴定人血浆Cohn IV级分中的103个N-糖基化位点。此外，Endo D/H消化独特地能够鉴定23个岩藻糖基化N-糖基化位点。还鉴定了几种O-糖基化肽，其中连接了单个N-乙酰己糖胺，可以认为是由于与Endo D/H一起使用的外切葡糖苷酶对O-聚糖结构进行了部分去糖基化。
Global proteome analysis of protein glycosylation is a major challenge due to the inherent heterogeneous and diverse nature of this post-translational modification. It is therefore common to enzymatically remove glycans attached to protein or peptide chains prior to mass spectrometric analysis, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide-N-glycosidase (PNGase) digestion, with concomitant deamidation of the released asparagine residue. The reaction is carried out in (H2O)-O-18 to facilitate identification of the formerly glycosylated peptide by incorporatation of O-18 into the formed aspartic acid residue. (2) Digestion with two endo-beta-N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond between the two N, acetylglucosamine (GIcNAc) residues in the conserved N-glycan core structure, leaving single GIcNAc residues with putative fucosyl side chains attached to the peptide. To enable digestion of complex and hybrid type N-glycans, a number of exoglycosidases (beta-galactosidase, neuraminidase and N-acetyl-beta-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides were also identified with a single N-acetylhexosamine attached, arguably due to partial deglycosylation of O-glycan structures by the exoglycosidases used together with Endo D/H.