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An improved evanescent fluorescence scanner suitable for high-resolution glycome map** of formalin-fixed paraffin-embedded tissue sections

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Abstract

Lectin microarray (LMA) is a high-throughput platform that enables the rapid and sensitive analysis of N- and O-glycans attached to glycoproteins in biological samples, including formalin-fixed paraffin-embedded (FFPE) tissue sections. Here, we evaluated the sensitivity of the advanced scanner based on the evanescent-field fluorescence principle, which is equipped with a 1× infinity correction optical system and a high-end complementary metal-oxide semiconductor (CMOS) image sensor in digital binning mode. Using various glycoprotein samples, we estimated that the mGSR1200-CMOS scanner has at least fourfold higher sensitivity for the lower limit of linearity range than that of a previous charge-coupled device scanner (mGSR1200). A subsequent sensitivity test using HEK293T cell lysates demonstrated that cell glycomic profiling could be performed with only three cells, which has the potential for the glycomic profiling of cell subpopulations. Thus, we examined its application in tissue glycome map**, as indicated in the online LM-GlycomeAtlas database. To achieve fine glycome map**, we refined the laser microdissection-assisted LMA procedure to analyze FFPE tissue sections. In this protocol, it was sufficient to collect 0.1 mm2 of each of the tissue fragments from 5-μm-thick sections, which differentiated the glycomic profile between the glomerulus and renal tubules of a normal mouse kidney. In conclusion, the improved LMA enables high-resolution spatial analysis, which expands the possibilities of its application classifying cell subpopulations in clinical FFPE tissue specimens. This will be used in the discovery phase for the development of novel glyco-biomarkers and therapeutic targets, and to expand the range of target diseases.

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Data availability

The data presented in this study are available on reasonable request from the corresponding author.

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Acknowledgements

We are grateful to Dr. Atsushi Matsuda and Dr. Takanori Wagatsuma of Keio University, Dr. Yoko Itakura of the Tokyo Metropolitan Institute of Gerontology, and Dr. Takahiro Hiono and Ms. Hiroko Shimazaki of AIST for their valuable discussions and comments.

Funding

This study was funded by a project for utilizing glycans in the development of innovative drug discovery technologies (grant number JP20ae0101021h0005) from the Japan Agency for Medical Research and Development (AMED).

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    Authors and Affiliations

    1. Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, 305-8565, Japan

      Patcharaporn Boottanun, Chiaki Nagai-Okatani, Misugi Nagai, Umbhorn Ungkulpasvich & Atsushi Kuno

    2. GlycoTechnica Ltd, 101 Hiranobiru3, 5-28-6 Utsukushigaoka, Aoba-Ku, Yokohama, Kanagawa, 225-0002, Japan

      Shinjiro Yamane

    3. EMUKK LLC, 2-21-19, Matsunoki, Kuwana, Mie, 511-0902, Japan

      Masao Yamada

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    1. Patcharaporn Boottanun
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    Correspondence to Chiaki Nagai-Okatani or Atsushi Kuno.

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    Institutional review board statement

    All animal experiments were performed in accordance with relevant guidelines and regulations. The animal study protocol was approved by the Institutional Animal Care and Use Committee at AIST (no. 2022-082; date of approval, 22/06/2022).

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    The authors declare no competing interests.

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    Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

    Published in the topical collection Recent Advances in Ultrasensitive Omics Techniques with guest editor Joseph Zaia.

    Patcharaporn Boottanun and Chiaki Nagai-Okatani contributed equally to this work.

    Shinjiro Yamane has retired from GlycoTechnica Ltd.

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    Boottanun, P., Nagai-Okatani, C., Nagai, M. et al. An improved evanescent fluorescence scanner suitable for high-resolution glycome map** of formalin-fixed paraffin-embedded tissue sections. Anal Bioanal Chem 415, 6975–6984 (2023). https://doi.org/10.1007/s00216-023-04824-2

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