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Spin-Labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells

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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.

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Funding

This study was supported by NIGMS/NIH (R35GM131686; ZG), NSF (MCB-1715384; GEF), an NIH instrumentation grant (S10 RR031603) for the Bruker E500 spectrometer, and Department of Chemistry, University of Florida for the Magnettech MS-5000 benchtop spectrometer. ZG thanks Steven and Rebecca Scott endowment to support our research.

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  1. Mohit Jaiswal and Trang T. Tran contribute equally to the current work.

Authors and Affiliations

  1. Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, FL, 32611, USA

    Mohit Jaiswal, Trang T. Tran, Jiatong Guo, Mingwei Zhou, Sayan Kundu, Zhongwu Guo & Gail E. Fanucci

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  1. Mohit Jaiswal
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  2. Trang T. Tran
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  3. Jiatong Guo
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  7. Gail E. Fanucci
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Contributions

MJ, JG, and SK contributed to molecule synthesis, cell culturing, glycoengineering, spin labeling and chemical fixation; TTT and MZ contributed to EPR studies and spectral fitting analyses; GEF and ZG were overall responsible for the project design and supervision. The manuscript was written through contributions of all authors, and all authors have given approval to the final version of the manuscript.

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Correspondence to Gail E. Fanucci.

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Jaiswal, M., Tran, T.T., Guo, J. et al. Spin-Labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells. Appl Magn Reson 55, 317–333 (2024). https://doi.org/10.1007/s00723-023-01624-w

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