Spin-Labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells
Spin-Labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells
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DOI：
10.1007/s00723-023-01624-w
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发表时间：
2023-10-09
影响因子：
1
通讯作者：
Fanucci，Gail E.
中科院分区：
文献类型：
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作者：
Jaiswal，Mohit;Tran，Trang T.;Fanucci，Gail E.
随着询问细胞上聚糖组织的新方法的发展，重要的是要在分子水平上了解化学固定如何影响结果和解释。定点自旋标记技术非常适合于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施加的那些。在这里，我们利用三种不同的含叠氮化物的糖与HeLa细胞的代谢聚糖工程，以纳入叠氮基聚糖，通过点击反应与基于DBCO的氮氧化物部分修饰。连续波X-波段电子顺磁共振光谱的化学固定和自旋标记的时间顺序的影响如何影响的局部流动性和可及性的氮氧自由基标记的聚糖在HeLa细胞的糖萼。结果表明，多聚甲醛化学固定可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，在分析数据时应谨慎。
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.