Mohit Jaiswal, Trang T. Tran, Jiatong Guo, Mingwei Zhou, Sayan Kundu, Zhongwu Guo, Gail E. Fanucci
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引用次数: 0
Abstract
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.
随着研究细胞聚糖组织的新方法的发展,从分子水平了解化学固定如何影响结果和解释是非常重要的。位点定向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。在这里,我们利用三种不同的含叠氮化物的糖类与 HeLa 细胞进行代谢聚糖工程,通过点击反应将叠氮聚糖与基于 DBCO 的亚硝基修饰在一起。连续波 X 波段电子顺磁共振光谱法用于描述化学固定和自旋标记的时间顺序如何影响硝基氧化物标记的聚糖在 HeLa 细胞糖萼中的局部流动性和可及性。结果表明,用多聚甲醛进行化学固定会改变局部聚糖的流动性,因此在进行化学固定和细胞标记的任何研究中分析数据时都应小心谨慎。
期刊介绍:
Applied Magnetic Resonance provides an international forum for the application of magnetic resonance in physics, chemistry, biology, medicine, geochemistry, ecology, engineering, and related fields.
The contents include articles with a strong emphasis on new applications, and on new experimental methods. Additional features include book reviews and Letters to the Editor.