Structural elucidation of O-linked glycopeptides by high energy collision-induced dissociation
Structural elucidation of O-linked glycopeptides by high energy collision-induced dissociation
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DOI：
10.1016/1044-0305(95)00682-6
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发表时间：
1996-04-01
影响因子：
3.2
通讯作者：
Hardy, MR
中科院分区：
文献类型：
--
作者：
Medzihradszky, KF;GilleceCastro, BL;Hardy, MR
通过高能碰撞诱导解离（CID）分析了具有和不具有唾液酸存在的GalNAc核心的O-连接糖肽。我们表明，糖基化前体离子的CID光谱包含足够的信息来识别肽序列并确定糖基化位点。研究了先前从牛胎球蛋白的胰蛋白酶消化物制备的去唾液酸O-连接糖肽。其中一种糖肽仅在Thr（262）处含有单个Hex（己糖）-HexNAc（N-乙酰己糖胺）取代，而另一种糖肽在Thr（262）和Ser（264）处均显示Hex-HexNAc部分。此外，从链霉蛋白酶消化的唾液酸和去唾液酸胎球蛋白糖肽衍生与叔丁氧羰基酪氨酸，并通过高能CID分析。通过使用内切-α-N-乙酰氨基半乳糖苷酶的底物特异性证实了Ser（264）处存在Gal β（1，3）GalNAc核心结构。这些研究揭示了在所用的内切-α-N-乙酰氨基半乳糖苷酶制剂中存在对β（1，4）键特异的β-半乳糖苷酶。最后，N-和O-糖基键的相对稳定性，以高能量CID的合成N-连接的糖肽与类似的O-连接的结构的行为的比较的基础上解决。
O-linked glycopeptides that bear a GalNAc core with and without the presence of sialic acid have been analyzed by high energy collision-induced dissociation (CID). We show that the CID spectra from the glycosylated precursor ions contain sufficient information to identify the peptide sequence and to determine the glycosylated site(s). Asialo O-linked glycopeptides, previously prepared from a tryptic digest of bovine fetuin were studied. One of the glycopeptides contained only a single Hex (hexose)-HexNAc (N-acetylhexosamine) substitution at Thr(262), whereas the other exhibited Hex-HexNAc moieties at both Thr(262) and Ser(264). In addition, sialo and asialo fetuin glycopeptides from a pronase digest were derivatized with t-butoxycarbonyl-tyrosine, and characterized by high energy CID analysis. The presence of a Gal beta(1,3)GalNAc core structure at Ser(264) was confirmed by using the substrate specificity of endo-alpha-N-acetylgalactosaminidase. These studies revealed the presence of a P-galactosidase specific for beta(1,4) linkages in the endo-alpha-N-acetylgalactosaminidase preparation employed. Finally, the relative stability of N- and O-glycosyl bonds to high energy CID is addressed based upon comparison of the behavior of a synthetic N-linked glycopeptide with analogous O-linked structures.