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Development of a highly efficient solubilization method for mass spectrometric analysis of phospholipids in living single cells

  • Special Issue: Original Paper
  • Novel Analytical Technologies Contributing to Clinical and Pharmaceutical Research Fields
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Abstract

Phospholipids are vital constituents of the cell membrane and aid in signal transduction. Phospholipid profiles vary distinctively with the cell type. Notably, specific phospholipid molecules are present in significantly higher or lower concentrations in cancer cells versus normal cells. In this study, live single-cell mass spectrometry (MS) was developed for analyzing phospholipids at the single-cell level. This method facilitates rapid molecular analysis of cells under microscopic observation. For nanoelectrospray ionization, phospholipids were extracted from single cells isolated in a glass capillary through a high-efficiency process. Cell-derived phosphatidylcholines were detected with high sensitivity when trehalose C14 was added as a solubilizing reagent. Trehalose C14 can solubilize cells at low concentrations owing to its low critical micelle concentration, and exerts minimal matrix effects (such as suppressing ionization and causing peak overlap) in the MS analysis of cellular molecules. Analyses of phospholipids in Raji and HEV0070 cells using the developed method revealed specific peaks of phosphatidylcholine and sphingomyelin in the respective cells. The developed technique not only affords phospholipid profiles at the single-cell level, but also holds promise for identifying biomarkers associated with various diseases, particularly cancer.

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Data availability

The data analyzed during the current study are available from the corresponding author on request.

References

  1. J.N. Veen, J.P. Kennelly, S. Wan, J.E. Vance, D.E. Vance, R.L. Jacobs, The critical role of phosphatidylcholine and phosphatidylethanolamine metabolism in health and disease. Biochim. Biophys. Acta. Biomembr. 1859, 1558–1572 (2017). https://doi.org/10.1016/j.bbamem.2017.04.006

    Article  CAS  PubMed  Google Scholar 

  2. F. F. Eiriksson, M. K. Nøhr, M. Costa, S. K. Bödvarsdottir, H. M. Ögmundsdottir, M. Thorsteinsdottir, Lipidomic study of cell lines reveals differences between breast cancer subtypes., PLoS One, 15, e0231289 (2020). **

  3. S.E. Hancock, E. Ding, E.J. Beves, T. Mitchell, N. Turner, FACS-assisted single-cell lipidome analysis of phosphatidylcholines and sphingomyelins in cells of different lineages. J. Lipid Res. 64, 100341 (2023). https://doi.org/10.1016/j.jlr.2023.100341

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. T.M. Domenick, E.L. Gill, V. Vedam-Mai, R.A. Yost, Mass spectrometry-based cellular metabolomics: current approaches, applications, and future directions. Anal. Chem. 93, 546–566 (2021). https://doi.org/10.1021/acs.analchem.0c04363

    Article  CAS  PubMed  Google Scholar 

  5. P. Dalerba, T. Kalisky, D. Sahoo, P. S. Rajendran, M. E. Rothenberg, A. A. Leyrat, S. Sim, J. Okamoto, D. M. Johnston, D. Qian, M. Zabala, J. Bueno, N. F. Neff, J. Wang, A. A. Shelton, B. Visser, S. Hisamori, Y. Shimono, M. de Wetering, H. Clevers, M. F. Clarke, S. R. Quake, Single-cell dissection of transcriptional heterogeneity in human colon tumors., Nature Biotechnology, 29, 1120-1127 (2011). 10.1038%2Fnbt.2038

  6. A. Biswas, S. De, Drivers of dynamic intratumor heterogeneity and phenotypic plasticity. Am. J. Physio. Cell Physiol. 320, C750–C760 (2021). https://doi.org/10.1152/ajpcell.00575.2020

    Article  CAS  Google Scholar 

  7. S. Shimma, Y. Sugiura, T. Hayasaka, Y. Hoshikawa, T. Noda, M. Setou, MALDI-based imaging mass spectrometry revealed abnormal distribution of phospholipids in colon cancer liver metastasis. J. Chromatogr. B 855, 98–103 (2007). https://doi.org/10.1016/j.jchromb.2007.02.037

    Article  CAS  Google Scholar 

  8. Y. Uchiyama, T. Hayasaka, N. Masaki, Y. Watanabe, K. Masumoto, T. Nagata, F. Katou, M. Setou, Imaging mass spectrometry distinguished the cancer and stromal regions of oral squamous cell carcinoma by visualizing phosphatidylcholine (16:0/16:1) and phosphatidylcholine (18:1/20:4). Anal. Bioanal. Chem. 406, 1307–1316 (2014). https://doi.org/10.1007/s00216-013-7062-3

    Article  CAS  PubMed  Google Scholar 

  9. K.C. O’Neill, E. Liapis, B.T. Harris, D.S. Perlin, C.L. Carter, Mass spectrometry imaging discriminates glioblastoma tumor cell subpopulations and different microvascular formations based on their lipid profiles. Sci. Rep. 12(17069), 6 (2022). https://doi.org/10.1038/s41598-022-22093-4

    Article  CAS  Google Scholar 

  10. H. Mizuno, N. Tsuyama, T. Harada, T. Masujima, Live single-cell video-mass spectrometry for cellular and subcellular molecular detection and cell classification. J. Mass Spectrom. 43, 1692–1700 (2008). https://doi.org/10.1002/jms.1460

    Article  CAS  PubMed  Google Scholar 

  11. T. Fujii, S. Matsuda, M.L. Tejedor, T. Esaki, I. Sakane, H. Mizuno, N. Tsuyama, T. Masujima, Direct metabolomics for plant cells by live single-cell mass spectrometry. Nat. Protoc. 10, 1445–1456 (2015). https://doi.org/10.1038/nprot.2015.084

    Article  CAS  PubMed  Google Scholar 

  12. K. Yahata, H. Mizuno, E. Sugiyama, K. Todoroki, Analysis of the intracellular localization of amiodarone using live single-cell mass spectrometry. J. Pharm. Biomed. Anal. 205, 114318 (2021). https://doi.org/10.1016/j.jpba.2021.114318

    Article  CAS  PubMed  Google Scholar 

  13. J.S. Behnke, L.H. Urner, Emergence of mass spectrometry detergents for membrane proteomics. Anal. Bioanal. Chem. 415, 3897–3909 (2023). https://doi.org/10.1007/s00216-023-04584-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. M. Rühl, S. Schönborn, M. Karas, Detergent-assisted sample preparation for MALDI-MS: Investigation of octylglucoside and docecylmaltoside for matrix crystallization, on-plate digestion, and trypsin activity. J. Mass Spectrom. 53, 675–679 (2018). https://doi.org/10.1002/jms.4203

    Article  CAS  PubMed  Google Scholar 

  15. M. Iwakawa, M. Goto, S. Noda, M. Sagara, S. Yamada, N. Yamamoto, Y. Kawakami, Y. Matsui, Y. Miyazawa, H. Yamazaki, H. Tsuji, T. Ohno, J. Mizoe, H. Tsujii, T. Imai, DNA repair capacity measured by high throughput alkaline comet assays in EBV-transformed cell lines and peripheral blood cells from cancer patients and healthy volunteers. Mutat. Res. 588, 1–6 (2005). https://doi.org/10.1016/j.mrgentox.2005.07.012

    Article  CAS  PubMed  Google Scholar 

  16. J. Xia, D.S. Wishart, Web-based inference of biological patterns, functions and pathways from metabolomic data using MetaboAnalyst. Nat. Protocols 6, 743–760 (2011). https://doi.org/10.1038/nprot.2011.319

    Article  CAS  PubMed  Google Scholar 

  17. H. Tao, Y. Fu, A. Thompson, S. C. Lee, N. Mahoney, R. C. Stevens, Q. Zhang, Synthesis and Properties of Dodecyl Trehaloside Detergents for Membrane Protein Studies., Langmuir, 28, 11173–11181 (2012). 10.1021%2Fla3020404

  18. N. Hasegawa, H. Jonotsuka, K. Miki, K. Takeda, X-ray structure analysis of bacteriorhodopsin at 1.3 A resolution. Sci. Rep. 8, 13123 (2018). https://doi.org/10.1038/s41598-018-31370-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. G. Wang, M. Qiu, X. Xing, J. Zhou, H. Yao, M. Li, R. Yin, Y. Hou, Y. Li, S. Pan, Y. Huang, F. Yang, F. Bai, H. Nie, S. Di, L. Guo, Z. Meng, J. Wang, Y. Yin, Lung cancer scRNA-seq and lipidomics reveal aberrant lipid metabolism for early-stage diagnosis, Sci. Transl. Med.. 14, eabk2756 (2022). https://doi.org/10.1126/scitranslmed.abk2756

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Acknowledgements

This study was supported in part of Grant-in-Aid for Scientific Research (19K07028 and 22K06551) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (H.M.).

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Authors and Affiliations

  1. School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-Ku, Shizuoka, Shizuoka, 422-8526, Japan

    Jo Sakata, Aogu Furusho, Eiji Sugiyama, Kenichiro Todoroki & Hajime Mizuno

  2. Central Research Institute, ITO EN, Ltd., 21 Mekami, Makinohara, Shizuoka, 421-0516, Japan

    Iwao Sakane

  3. Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-Ku, Nagoya, Aichi, 468-8503, Japan

    Hajime Mizuno

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  1. Jo Sakata
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  2. Aogu Furusho
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  3. Eiji Sugiyama
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  4. Iwao Sakane
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  5. Kenichiro Todoroki
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Contributions

JS, AF, and HM performed the experiments and analyzed the data. HM and AF contributed to experimental design. ES and IS contributed to analyzing data. KT and HM supervised this work. The manuscript was written by HM in collaboration with the other authors. All authors have approved the final version of the manuscript.

Corresponding author

Correspondence to Hajime Mizuno.

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Sakata, J., Furusho, A., Sugiyama, E. et al. Development of a highly efficient solubilization method for mass spectrometric analysis of phospholipids in living single cells. ANAL. SCI. 40, 917–924 (2024). https://doi.org/10.1007/s44211-024-00542-6

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  • DOI: https://doi.org/10.1007/s44211-024-00542-6

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