The Glycomics Center, Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824, USA.
Anal Bioanal Chem. 2019 Aug;411(20):5033-5045. doi: 10.1007/s00216-019-01899-8. Epub 2019 Jun 1.
Glycosaminoglycans (GAGs) are carbohydrate polyionic polymers that participate in a host of critically important biological processes. A significant difficulty in the comprehensive structural characterization of GAGs is the determination of specific sulfation position isomers. We chose to circumvent sulfate lability by its liberation followed by specific isotope exchange that makes it amenable to methylation, collisional induced dissociation, and MS disassembly for a detailed structural characterization. A set of chemistries that include sulfate release, isotopic (CD- and CD-CO-) replacement, and methylation have been modified to yield a stable product ideal for sequencing by MS. Disassembly of these samples provides a detailed read-out of sequence inclusive of all sulfation sites. As documenting steps, we applied these chemical modifications to a series of disaccharides and a synthetic GAG pentamer, Arixtra®. Upon disassembly, glycosidic and cross-ring cleavages define the monomer composition including individual sulfation positions. The N- and O-sulfates are differentiated by deuterium-containing mass compositions. The uronic methylesters do not significantly alter the fragmentation patterns. A fragment library of these products is being assembled as an adjunct to our larger fragment library, some 15 years in the making.
糖胺聚糖（GAGs）是参与许多重要生物过程的多糖聚阴离子聚合物。在全面结构表征 GAGs 时，一个显著的困难是确定特定硫酸化位置异构体。我们选择通过释放硫酸盐并进行特定的同位素交换来规避硫酸盐的不稳定性，这使得 GAGs 能够进行甲基化、碰撞诱导解离和 MS 解组装，从而进行详细的结构表征。一组包括硫酸盐释放、同位素（CD-和 CD-CO-）替换和甲基化的化学方法已经被修改，以产生适合 MS 测序的稳定产物。对这些样品的解组装提供了包括所有硫酸化位点的序列的详细读数。作为记录步骤，我们将这些化学修饰应用于一系列二糖和合成 GAG 五聚体 Arixtra®。在解组装后，糖苷键和交联环裂解定义了单体组成，包括各个硫酸化位置。N-和 O-硫酸盐通过含有氘的质量组成来区分。糖醛酸甲酯基不会显著改变片段模式。这些产物的片段库正在作为我们更大的片段库的辅助库进行组装，该库已经存在了 15 年。
