Differentiation of glycosphingolipid-derived glycan structural isomers by liquid chromatography/mass spectrometry
Differentiation of glycosphingolipid-derived glycan structural isomers by liquid chromatography/mass spectrometry
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DOI：
10.1093/glycob/cwq070
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发表时间：
2010-09-01
期刊：
影响因子：
4.3
通讯作者：
Teneberg, Susann
中科院分区：
文献类型：
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作者：
Karlsson, Hasse;Halim, Adnan;Teneberg, Susann
鞘糖脂的分离和表征在糖生物学的许多方面都很重要，但由于这些化合物中存在高度的异质性和异构性而难以实现。在这项研究中，从非酸性鞘糖脂通过酶消化与神经酰胺内切酶II的红球菌属的寡糖进行了分析，液相色谱/电喷雾电离质谱使用石墨化碳柱。实现了异构寡糖的解析，并且MS 2分析给出了完整的序列信息，并允许区分连接位置。先前已经描述了C-4上取代的GlcNAc和4-取代的Glc的诊断性交叉环（0，2）A型片段。诊断性交叉环（0，2）A型片段存在于H 2型（Fuc α 2Gal β 4GlcNAc β 4Gal β 4Glc）五糖的MS 2谱中，但不存在于H 1型五糖（Fuc α 2Gal β 3GlcNAc β 4Gal β 4Glc）的MS 2谱中。还从鞘糖脂衍生寡糖的还原端的4-取代的Glc获得了交叉环（0，2）A型片段。还在石墨化碳柱上色谱分离了球状系列（球状三糖（Gal α 4Gal β 4Glc）和球状四糖（GalNAc β 3Gal α 4Gal β 4Glc））和异球状系列（异球状三糖（Gal α 3Gal β 4Glc）和异球状四糖（GalNAc β 3Gal α 3Gal β 4Glc））的寡糖。此外，诊断碎片离子从交叉环（0，2）A-型裂解存在于MS 2光谱的globo系列寡糖，具有Gal取代的C-4。使用来自人胃上皮的非酸性鞘糖脂部分和来自8 × 10（7）骨髓来源的小鼠树突状细胞的部分纯化的非酸性鞘糖脂部分证明了该方法对组织来源样品的适用性。在这里，通过对神经酰胺内切酶释放的低聚糖进行液相色谱/质谱分析，可以初步鉴定从三糖神经酰胺到九糖神经酰胺的多种鞘糖脂。
Isolation and characterization of glycosphingolipids is of importance in many aspects of glycobiology, but is difficult to achieve due to the high degree of heterogeneity and isomerism present in these compounds. In this study, oligosaccharides obtained from non-acid glycosphingolipids by enzymatic digestion with endoglycoceramidase II of Rhodococcus sp. were analyzed by liquid chromatography/electrospray ionization mass spectrometry using graphitized carbon columns. Resolution of isomeric oligosaccharides was achieved, and the MS2 analyses gave complete sequence information and allowed differentiation of linkage positions. Diagnostic cross-ring (0,2)A-type fragments have previously been described for GlcNAc substituted on C-4 and for 4-substituted Glc. Diagnostic cross-ring (0,2)A-type fragments were present in the MS2 spectrum of the H type 2 (Fuc alpha 2Gal beta 4GlcNAc beta 4Gal beta 4Glc) pentasaccharide, but not in the MS2 spectrum of H type 1 pentasaccharide (Fuc alpha 2Gal beta 3GlcNAc beta 4Gal beta 4Glc). Cross-ring (0,2)A-type fragments were also obtained from the 4-substituted Glc at the reducing end of the glycosphingolipid-derived oligosaccharides. Oligosaccharides of the globo-series (globotriaose (Gal alpha 4Gal beta 4Glc) and globotetraose (GalNAc beta 3Gal alpha 4Gal beta 4Glc)) and the isoglobo-series (isoglobotriaose (Gal alpha 3Gal beta 4Glc) and isoglobotetraose (GalNAc beta 3Gal alpha 3Gal beta 4Glc)) were also chromatographically resolved on the graphitized carbon column. Furthermore, diagnostic fragment ions from cross-ring (0,2)A-type cleavages were present in the MS2 spectra of the globo-series oligosaccharides, having a Gal substituted on C-4. The applicability of this method on tissue-derived samples was demonstrated using a non-acid glycosphingolipid fraction from human gastric epithelium and a partially purified non-acid glycosphingolipid fraction from 8 x 10(7) bone marrow-derived mouse dendritic cells. Here, liquid chromatography/mass spectrometry of the oligosaccharides released by endoglycoceramidase allowed tentative identification of a number of glycosphingolipids ranging from tri- to nonaglycosylceramides.