Rouse J C, Strang A M, Yu W, Vath J E
Genetics Institute, Inc., Andover, Massachusetts 01810, USA.
Anal Biochem. 1998 Feb 1;256(1):33-46. doi: 10.1006/abio.1997.2450.
Matrix-assisted laser desorption-ionization (MALDI)-postsource decay (PSD) was used to differentiate glycoprotein-released N-linked oligosaccharide isomers directly from aliquots of glycosidase digests and peak fractions collected from high-pH anion exchange chromatography (HPAEC) with minimal sample handling and material. With the implementation of instrumental tuning and acquisition controls, MALDI-PSD of NMR-characterized high-mannose, hybrid, and complex standards resulted in spectra with reproducible fragment ion peak intensity ratios which correlated well to the respective oligosaccharide branching patterns. A "knowledge-based" strategy was utilized to characterize unknown isomeric N-glycan structures in which specific fragment ion types and their distributions in the unknown PSD spectrum were compared to those in PSD spectra of standards possessing similar structural features. This PSD knowledge-based isomeric differentiation strategy was applied to distinguishing recombinant glycoprotein-derived Man7 D1 versus D2/D3 isomers directly from a PNGaseF digest aliquot of high-mannose N-glycans based on branching differences. A precursor ion selection device was employed to isolate the component of interest from the mass profile without additional chromatographic isolation steps. MALDI-MS signal-to-background was maximized for direct PSD with on-the-probe sample clean-up methods. The asialo complex N-glycan PSD knowledge base was used to differentiate HPAEC peak fractions containing the tri- and tri'-antennary branching isomers and two tetraantennary isomers with antennal versus core fucose locations. Although the particular asialo complex N-glycan isomers here were well separated by HPAEC, MALDI-MS alerted us to their presence using m/z-derived monosaccharide compositions and PSD fragmentation allowed us to differentiate these structures using the HPAEC elution positions as guides.
基质辅助激光解吸电离（MALDI）-源后衰变（PSD）被用于直接从糖苷酶消化物的等分试样以及从高pH阴离子交换色谱（HPAEC）收集的峰馏分中区分糖蛋白释放的N-连接寡糖异构体，所需的样品处理和材料最少。通过实施仪器调谐和采集控制，对经核磁共振表征的高甘露糖型、杂合型和复合型标准品进行MALDI-PSD分析，得到的光谱具有可重现的碎片离子峰强度比，与各自的寡糖分支模式相关性良好。采用“基于知识”的策略来表征未知的N-聚糖异构体结构，即将未知PSD光谱中特定的碎片离子类型及其分布与具有相似结构特征的标准品的PSD光谱进行比较。这种基于PSD知识的异构体区分策略被应用于直接从高甘露糖型N-聚糖经肽:N-糖苷酶F消化的等分试样中区分重组糖蛋白衍生的Man7 D1与D2/D3异构体，依据的是分支差异。使用前体离子选择装置从质量分布图中分离出感兴趣的组分，无需额外的色谱分离步骤。通过探针上样品净化方法，使MALDI-MS的信号背景比在直接PSD分析中达到最大化。利用去唾液酸复合N-聚糖PSD知识库来区分含有三分支和三'分支异构体以及两种带有触角型和核心岩藻糖位置不同的四分支异构体的HPAEC峰馏分。尽管此处特定的去唾液酸复合N-聚糖异构体通过HPAEC得到了很好的分离，但MALDI-MS利用m/z衍生的单糖组成提醒我们它们的存在，而PSD碎片化使我们能够以HPAEC洗脱位置为指导来区分这些结构。
