Tools for glycoproteomic analysis: Size exclusion chromatography facilitates identification of tryptic glycopeptides with N-linked glycosylation sites
Tools for glycoproteomic analysis: Size exclusion chromatography facilitates identification of tryptic glycopeptides with N-linked glycosylation sites
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DOI：
10.1021/pr050275j
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发表时间：
2006-03-01
影响因子：
4.4
通讯作者：
Pierce, M
中科院分区：
文献类型：
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作者：
Alvarez-Manilla, G;Atwood, J;Pierce, M
蛋白质组学技术，如高效液相色谱-串联质谱仪(LC-MS/MS)，已被证明对鉴定糖蛋白上的特定糖基化位点(糖蛋白组学)是有用的。然而，通过复杂的糖蛋白混合物的胰酶作用产生的糖肽上的糖基化位点尤其难以识别，这既是因为一组糖链可能在特定的糖基化位点表达，又因为糖肽通常以相对较低的丰度存在(与非糖基化多肽相比，在多肽混合物中的丰度相对较低(2%至51/0)。以前报道的有助于糖肽鉴定的方法需要几个预富集步骤，涉及复杂的衍生化过程，或者仅限于存在于糖蛋白混合物中的所有糖结构的子集。由于在胰酶糖肽上表达的N-糖链对它们的质量有很大贡献，我们证明尺寸排除层析(SEC)提供了相对于非糖基化多肽的显著的N-链糖链的富集物。经PNGase-F处理后，经LC-MS/MS鉴定，糖基化多肽的单同位素质量增加了0.984 Da。对人血清进行的分析表明，这种SEC糖肽分离程序使LC-MS/MS鉴定的糖肽总数至少增加了3倍，表明这种简单、非选择性、快速的方法也是一种有效的方法[有助于鉴定具有N-连接糖基化位点的多肽]。
Proteomic techniques, such as HPLC coupled to tandem mass spectrometry (LC-MS/MS), have proved useful for the identification of specific glycosylation sites on glycoproteins (glycoproteomics). Glycosylation sites on glycopeptides produced by trypsinization of complex glycoprotein Mixtures, however, are particularly difficult to identify both because a repertoire of glycans may be expressed at a particular glycosylation site, and because glycopeptides are usually present in relatively low abundance (2% to 51/0 in peptide mixtures compared to nonglycosylated peptides. Previously reported methods to facilitate glycopeptide identification require either several pre-enrichment steps, involve complex derivatization procedures, or are restricted to a subset of all the glycan structures that are present in a glycoprotein mixture. Because the N-linked glycans expressed on tryptic glycopeptides contribute substantially to their mass, we demonstrate that size exclusion chromatography (SEC) provided a significant enrichment of N-linked glycopeptides relative to nonglycosylated peptides. The glycosylated peptides were then identified by LC-MS/MS after treatment with PNGase-F by the monoisotopic mass increase of 0.984 Da caused by the deglycosylation of the peptide. Analyses performed on human serum showed that this SEC glycopeptide isolation procedure results in at least a 3-fold increase in the total number of glycopeptides identified by LC-MS/MS, demonstrating that this simple, nonselective, rapid method is an effective too[ to facilitate the identification of peptides with N-linked glycosylation sites.