Analysis of Invertebrate and Protist N-Glycans.
Analysis of Invertebrate and Protist N-Glycans.
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无脊椎动物和原生n-聚糖的分析。
DOI：
10.1007/978-1-4939-6493-2_13
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Wilson, Iain B H
中科院分区：
文献类型：
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作者：
Hykollari, Alba;Paschinger, Katharina;Eckmair, Barbara;Wilson, Iain B H
来自无脊椎动物和原生生物的N-聚糖通常具有不寻常的结构，这给分析带来了挑战。核心和触角修饰都可以与更熟悉的脊椎动物聚糖基序完全不同;因此，与“简单”生物具有“简单”N-聚糖的概念相反，在一系列物种中发现了相当复杂的寡糖结构，包括两性离子和阴离子结构。因此，为了便于最大可能范围的结构的优化解析，这些生物体的糖组学分析工作流程应包括顺序释放和分馏步骤。肽：N-糖苷酶F足以从真菌和一些原生生物中分离N-聚糖，但在大多数无脊椎动物中存在核心α 1，3-岩藻糖，因此需要通过肽：N-糖苷酶A从糖肽中释放聚糖。随后使用石墨化碳和反相树脂进行固相萃取，可在高压液相色谱（HPLC）和基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）之前分离不同类别的N-聚糖。根据存在的聚糖的类型和数量，反相或正相HPLC（或两者串联）甚至可以在MALDI-TOF MS和MS/MS之前分离单个异构体或同量异位结构。使用酶或化学处理可以获得进一步的见解，尽管一些聚糖修饰（特别是甲基化）是抗性的。使用一系列方法，有时可以分配来自单个生物体的多达100个结构，这种复杂性引发了关于这些聚糖功能的进化问题。
N-glycans from invertebrates and protists have often unusual structures which present analytical challenges. Both core and antennal modifications can be quite different from the more familiar vertebrate glycan motifs; thereby, contrary to the concept that ‘simple’ organisms have ‘simple’ N-glycans, rather complex oligosaccharides structures, including zwitterionic and anionic ones, have been found in a range of species. Thus, to facilitate the optimised elucidation of the maximal possible range of structures, the analytical workflow for glycomics of these organisms should include sequential release and fractionation steps. Peptide:N-glycosidase F is sufficient to isolate N-glycans from fungi and some protists, but in most invertebrates core α1,3-fucose is present, so release of the glycans from glycopeptides by peptide:N-glycosidases A is required. Subsequent solid-phase extraction with graphitised carbon and reversed phase resins enables different classes of N-glycans to be separated prior to high-pressure liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Depending on the types and numbers of glycans present, either reversed- or normal-phase HPLC (or both in series) enable even single isomeric or isobaric structures to be separated prior to MALDI-TOF MS and MS/MS. The use of enzymatic or chemical treatments allows further insights to be gained, although some glycan modifications (especially methylation) are resistant. Using a battery of methods, sometimes up to 100 structures from a single organism can be assigned, a complexity which raises evolutionary questions regarding the function of these glycans.