Nuffield Department of Medicine, Target Discovery Institute, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom.
Centre for Biological Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, Life Sciences Building 85, Highfield Campus, Southampton, SO17 1BJ, United Kingdom.
Mass Spectrom Rev. 2020 Sep;39(5-6):586-679. doi: 10.1002/mas.21622. Epub 2020 Apr 24.
N-glycans from glycoproteins are complex, branched structures whose structural determination presents many analytical problems. Mass spectrometry, usually conducted in positive ion mode, often requires extensive sample manipulation, usually by derivatization such as permethylation, to provide the necessary structure-revealing fragment ions. The newer but, so far, lesser used negative ion techniques, on the contrary, provide a wealth of structural information not present in positive ion spectra that greatly simplify the analysis of these compounds and can usually be conducted without the need for derivatization. This review describes the use of negative ion mass spectrometry for the structural analysis of N-linked glycans and emphasises the many advantages that can be gained by this mode of operation. Biosynthesis and structures of the compounds are described followed by methods for release of the glycans from the protein. Methods for ionization are discussed with emphasis on matrix-assisted laser desorption/ionization (MALDI) and methods for producing negative ions from neutral compounds. Acidic glycans naturally give deprotonated species under most ionization conditions. Fragmentation of negative ions is discussed next with particular reference to those ions that are diagnostic for specific features such as the branching topology of the glycans and substitution positions of moieties such as fucose and sulfate, features that are often difficult to identify easily by conventional techniques such as positive ion fragmentation and exoglycosidase digestions. The advantages of negative over positive ions for this structural work are emphasised with an example of a series of glycans where all other methods failed to produce a structure. Fragmentation of derivatized glycans is discussed next, both with respect to derivatives at the reducing terminus of the molecules, and to methods for neutralization of the acidic groups on sialic acids to both stabilize them for MALDI analysis and to produce the diagnostic fragments seen with the neutral glycans. The use of ion mobility, combined with conventional mass spectrometry is described with emphasis on its use to extract clean glycan spectra both before and after fragmentation, to separate isomers and its use to extract additional information from separated fragment ions. A section on applications follows with examples of the identification of novel structures from lower organisms and tables listing the use of negative ions for structural identification of specific glycoproteins, glycans from viruses and uses in the biopharmaceutical industry and in medicine. The review concludes with a summary of the advantages and disadvantages of the technique. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.
糖蛋白中的 N-糖链是复杂的分支结构，其结构测定存在许多分析问题。质谱通常在正离子模式下进行，通常需要广泛的样品处理，通常通过衍生化（如全甲基化）来提供必需的揭示结构的碎片离子。较新但到目前为止使用较少的负离子技术则相反，提供了大量正离子光谱中不存在的结构信息，极大地简化了这些化合物的分析，并且通常无需衍生化即可进行。本文综述了负离子质谱在 N-连接糖链结构分析中的应用，并强调了这种操作模式带来的许多优势。本文首先描述了化合物的生物合成和结构，然后介绍了从蛋白质中释放糖链的方法。本文还讨论了离子化方法，重点介绍基质辅助激光解吸/电离（MALDI）以及从中性化合物产生负离子的方法。在大多数电离条件下，酸性糖通常会给出去质子化的物质。接下来讨论了负离子的碎片化，特别提到了那些对糖链分支拓扑结构以及诸如岩藻糖和硫酸盐等基团取代位置等特定特征具有诊断意义的离子，这些特征通常很难通过常规技术（如正离子碎片化和外切糖苷酶消化）轻松识别。通过一个系列糖的例子强调了负离子相对于正离子的结构优势，在这个例子中，所有其他方法都未能产生结构。接下来讨论了衍生糖链的碎片化，既讨论了分子还原末端的衍生物，也讨论了中和唾液酸上酸性基团的方法，既可以稳定它们进行 MALDI 分析，又可以产生与中性糖链相同的诊断片段。接下来描述了离子淌度与常规质谱的联用，重点介绍了其在碎片前和碎片后提取清洁糖谱、分离异构体以及从分离的片段离子中提取附加信息的用途。随后的应用部分介绍了从低等生物中鉴定新结构的例子，以及列出了负离子用于结构鉴定特定糖蛋白、病毒糖链以及在生物制药行业和医学中的用途的表格。本文以总结该技术的优缺点作为结尾。© 2020 约翰威立父子有限公司。质谱评论。
