Department of Molecular Sciences, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O. Box 7015, SE-750 07, Uppsala, Sweden.
Hirszfeld Institute of Immunology and Experimental Therapy, 53-114, Wroclaw, Poland.
Carbohydr Res. 2023 Sep;531:108888. doi: 10.1016/j.carres.2023.108888. Epub 2023 Jun 24.
Hyaluronan (HA), a member of the GAG family of glycans, has many diverse biological functions that vary a lot depending on the length of the HA chain and its concentration. A better understanding of the structure of different-sized HA at the atomic level is therefore crucial to decipher these biological functions. NMR is a method of choice for conformational studies of biomolecules, but there are limitations due to the low natural abundance of the NMR active nuclei C and N. We describe here the metabolic labeling of HA using the bacterium Streptococcus equi subsp. Zooepidemicus and the subsequent analysis by NMR and mass spectrometry. The level of C and N isotope enrichment at each position was determined quantitatively by NMR spectroscopy and was further confirmed by high-resolution mass spectrometry analysis. This study provides a valid methodological approach that can be applied to the quantitative assessment of isotopically labeled glycans and will help improve detection capabilities and facilitate future structure-function relationship analysis of complex glycans.
透明质酸（HA）是糖胺聚糖（GAG）家族的成员，具有许多不同的生物学功能，这些功能因 HA 链的长度和浓度而异。因此，更好地了解不同大小的 HA 在原子水平上的结构对于破译这些生物学功能至关重要。NMR 是生物分子构象研究的首选方法，但由于 NMR 活性核 C 和 N 的天然丰度低，存在一定的局限性。我们在这里描述了使用马链球菌兽亚种（Streptococcus equi subsp. Zooepidemicus）对 HA 进行代谢标记，以及随后通过 NMR 和质谱分析。通过 NMR 光谱定量确定了每个位置的 C 和 N 同位素丰度，并通过高分辨率质谱分析进一步确认。这项研究提供了一种有效的方法，可以应用于对同位素标记聚糖的定量评估，并有助于提高检测能力，为未来复杂聚糖的结构-功能关系分析提供便利。
