As new methods to interrogate glycan organization on cells develop, it is important to have molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction. Continuous wave X-band electron paramagnetic resonance spectroscopy is employed to characterize how the chronological sequence of chemical fixation and spin labeling impacts the local mobility and accessibility of the nitroxide-labeled glycans in the glycocalyx of HeLa cells. Results demonstrate that chemical fixation with paraformaldehyde can alter local glycan mobility and care should be taken in the analysis of data in any study where chemical fixation and cellular labeling occur.

随着研究细胞上聚糖组织的新方法的发展，在分子水平上了解化学固定如何影响结果和解释非常重要。定点自旋标记技术非常适合研究自旋标记迁移率如何受到局部环境条件的影响，例如多聚甲醛细胞固定方法的交联效应所造成的影响。在这里，我们利用三种不同的含叠氮糖对 HeLa 细胞进行代谢聚糖工程，以掺入叠氮聚糖，这些聚糖通过点击反应用基于 DBCO 的硝基氧部分进行修饰。采用连续波 X 波段电子顺磁共振波谱来表征化学固定和自旋标记的时间顺序如何影响 HeLa 细胞糖萼中硝基氧标记聚糖的局部迁移性和可及性。结果表明，用多聚甲醛进行化学固定可以改变局部聚糖的流动性，在任何发生化学固定和细胞标记的研究中，在数据分析时应小心谨慎。