AbstractGlycans constitute a significant fraction of biomolecular diversity on cellular surfaces across all kingdoms of life. As the structure of glycans is not directly encoded by the organism’s DNA, it is impossible to use high-throughput DNA technologies to study the role of cellular glycosylation or to understand how glycocalyx is recognized by glycan-binding proteins (GBPs).

摘要聚糖构成了所有生命王国细胞表面生物分子多样性的重要组成部分。由于聚糖的结构不是由生物体的DNA直接编码的，因此不可能使用高通量DNA技术来研究细胞糖基化的作用或了解糖萼如何被聚糖结合蛋白（GBP）识别。

To address this gap, we recently described a liquid glycan array (LiGA) platform that allows profiling of glycan–GBP interactions on the surface of live cells in vitro and in vivo using next-generation sequencing. LiGA is a library of DNA-barcoded bacteriophages, where each clonal bacteriophage displays 5–1,500 copies of a glycan and the distinct DNA barcode inside each bacteriophage clone encodes the structure and density of the displayed glycans.

为了解决这一差距，我们最近描述了一种液体聚糖阵列（LiGA）平台，该平台可以使用下一代测序技术在体外和体内分析活细胞表面的聚糖-GBP相互作用。LiGA是DNA条形码噬菌体的文库，其中每个克隆噬菌体显示5-1500个聚糖拷贝，每个噬菌体克隆内不同的DNA条形码编码所显示聚糖的结构和密度。

Deep sequencing of the glycophages associated with live cells yields a glycan-binding profile of GBPs expressed on the surface of cells. This protocol provides detailed instructions for how to use LiGA to probe cell surface receptors and includes information on the preparation of glycophages, analysis by MALDI–TOF mass spectrometry, the assembly of a LiGA library and its deep sequencing.

与活细胞相关的糖噬菌体的深度测序产生在细胞表面表达的GBP的聚糖结合谱。该协议提供了如何使用LiGA探测细胞表面受体的详细说明，并包括有关糖噬菌体制备，MALDI-TOF质谱分析，LiGA文库组装及其深度测序的信息。

Using this protocol, we measure glycan-binding profiles of the immunomodulatory sialic acid-binding immunoglobulin-like lectins‑1, -2, -6, -7 and -9 expressed on the surface of different cell types. Compared with existing methods that require complex specialist equipment, this method allows users with basic molecular biology expertise to measure the precise glycan-binding profile of GBPs on the surface of any cell type expressing exogenous GBP within 2–3 d.Key points.

使用该方案，我们测量了在不同细胞类型表面表达的免疫调节性唾液酸结合免疫球蛋白样凝集素-1，-2，-6，-7和-9的聚糖结合谱。与需要复杂专业设备的现有方法相比，该方法允许具有基本分子生物学专业知识的用户在2-3天内测量表达外源性GBP的任何细胞类型表面上GBP的精确聚糖结合谱。要点。

This protocol describes the preparation of a liquid glycan array (LiGA) platform, a library of DNA-barcoded bacteriophages displaying 5–1,500 copies of a glycan. Deep sequencing of the glycophages associated with live cells yields a glycan-binding profile of GBPs displayed on the surface of cells.

该方案描述了液体聚糖阵列（LiGA）平台的制备，该平台是一个DNA条形码噬菌体文库，显示5-1500个聚糖拷贝。与活细胞相关的糖噬菌体的深度测序产生显示在细胞表面的GBP的聚糖结合谱。

The development of this technology enables testing of the biological role of multivalent glycan–lectin interactions in a multiplexed fashion, which was not previously possible.

该技术的发展使得能够以多重方式测试多价聚糖-凝集素相互作用的生物学作用，这在以前是不可能的。

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Fig. 1: Workflow for cell-surface binding assay using LiGA.Fig. 2: The workflow for PCR, deep-seq and bioinformatic analysis.Fig. 3: Siglec-7 enrichment profile on different cell milieu.Fig. 4: LiGA measures the binding specificity of Siglec-7, -9 and -2 on distinct cell surfaces.Fig. 5: LiGA profiling of Siglec-1 displayed on CHO cells..

图1：使用LiGA进行细胞表面结合测定的工作流程。图2:PCR，deep-seq和生物信息学分析的工作流程。图3：不同细胞环境下的Siglec-7富集曲线。图4:LiGA测量Siglec-7，-9和-2在不同细胞表面的结合特异性。图5:CHO细胞上显示的Siglec-1的LiGA分析。。

Data availability

数据可用性

All data relating to differential enrichment analysis are submitted as source data. All raw deep-sequencing data are publicly available in a searchable format on https://48hd.cloud/; for example, search ‘EF Sig9 jurkat’ to obtain raw sequencing data of LiGA EF post panning against Siglec-9+ Jurkat cells.

所有与差异富集分析有关的数据均作为源数据提交。所有原始深度测序数据都可以在https://48hd.cloud/；例如，搜索“EF Sig9 jurkat”以获得针对Siglec-9+jurkat细胞的LiGA EF后淘洗的原始测序数据。

Additionally, raw deep-sequencing data can also be requested from the corresponding author. DNA sequences of the three LiGA phage constructs with the reporter genes LacZ, mNeonGreen and mCherry have been deposited to GenBank (MN865131, MN865132, MN872303). Source data are provided with this paper..

此外，还可以从相应的作者那里请求原始的深度测序数据。具有报告基因LacZ，mNeonGreen和mCherry的三种LiGA噬菌体构建体的DNA序列已保存到GenBank（MN865131，MN865132，MN872303）。本文提供了源数据。。

Code availability

代码可用性

MATLAB and R scripts used to generate MALDI–TOF spectra and differential enrichment analysis, respectively, have been deposited to https://github.com/derdalab/liga.

分别用于生成MALDI-TOF光谱和差异富集分析的MATLAB和R脚本已保存到https://github.com/derdalab/liga.

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Download referencesAcknowledgementsWe thank the staff at the University of Alberta mass spectrometry facility (chemistry department) for help with MALDI analysis and S. Dang at the molecular biology service unit for assistance with Illumina sequencing. We acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) (RGPIN-2018-03815 to M.S.M.

下载参考文献致谢我们感谢阿尔伯塔大学质谱设施（化学系）的工作人员对MALDI分析的帮助，以及分子生物学服务部门的S.Dang对Illumina测序的帮助。我们感谢加拿大自然科学与工程研究委员会（NSERC）（RGPIN-2018-03815 to M.S.M.）的资助。

and RGPIN-2016-402511 to R.D.), the NSERC Accelerator Supplement (to R.D.), NSERC (RGPIN-2022-04484 to R.D.), Canadian Institutes of Health Research (CIHR) (no. 180445 to R.D.), GlycoNet (CR-29 and TP−22 to R.D.), and the Alberta Innovates Strategic Research Project to R.D. Infrastructure support was provided by the Canada Foundation for Innovation New Leader Opportunity (to R.D.

和RGPIN-2016-402511至R.D.）、NSERC加速器补充（至R.D.）、NSERC（RGPIN-2022-04484至R.D.）、加拿大卫生研究院（CIHR）（R.D.编号180445）、GlycoNet（R.D.的CR-29和TP-22）和艾伯塔省创新战略研究项目至R.D。基础设施支持由加拿大创新基金会新领导者机会（至R.D.）提供。

and M.S.M.). Many compounds were prepared by the Consortium for Functional Glycomics, supported by National Institutes of Health (NIH) GM061126. G.M.L. acknowledges funding from Alberta Innovates Graduate Student Scholarship.Author informationAuthors and AffiliationsDepartment of Chemistry, University of Alberta, Edmonton, Alberta, CanadaMirat Sojitra, Edward N.

和M.S.M.）。许多化合物是由美国国立卫生研究院（NIH）GM061126支持的功能糖组学联盟制备的。G、 M.L.感谢艾伯塔省创新研究生奖学金的资助。作者信息作者和附属机构艾伯塔大学化学系，艾伯塔省埃德蒙顿，加拿大达米拉特·索吉特拉，爱德华·N。

Schmidt, Guilherme M. Lima, Eric J. Carpenter, Kelli A. McCord, Alexey Atrazhev, Matthew S. Macauley & Ratmir DerdaDepartment of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, CanadaMatthew S. MacauleyNeuroscience and Mental Health Institute, University of Alberta, Edmonton, CanadaMatthew S.

。

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PubMed Google ScholarContributionsM.S. performed modifications of phages by glycans, MALDI analysis and cell binding assays. G.M.L. and A.A. optimized the cell binding assay. E.N.S., K.A.M. and M.S.M. generated the Siglec expressing cell lines. M.S., E.J.C. and R.D. performed statistical analysis and wrote software in R and MATLAB.

PubMed谷歌学术贡献。S、 通过聚糖，MALDI分析和细胞结合测定对噬菌体进行修饰。G、 M.L.和A.A.优化了细胞结合测定。E、 N.S.，K.A.M.和M.S.M.产生了表达Siglec的细胞系。M、 S.，E.J.C.和R.D.进行了统计分析，并用R和MATLAB编写了软件。

R.D. and M.S. wrote the manuscript. R.D. and M.S.M. edited the final manuscript and contributed intellectual and strategic input. All authors approved the final manuscript.Corresponding authorCorrespondence to.

R、 D.和M.S.写了手稿。R、 D.和M.S.M.编辑了最终手稿，并提供了知识和战略投入。所有作者都批准了最终稿件。对应作者对应。

Ratmir Derda.Ethics declarations

拉特米尔·德尔达。道德宣言

Competing interests

相互竞争的利益

R.D. is a shareholder of the start-up company 48Hour Discovery Inc. that licensed the patent application (WO2018141058A1) describing LiGA technology. The other authors declare no competing interests.

R、 D.是初创公司48Hour Discovery Inc.的股东，该公司授权了描述LiGA技术的专利申请（WO2018141058A1）。其他作者声明没有利益冲突。

Peer review

同行评审

Peer review information

同行评审信息

Nature Protocols thanks Zheng Li, Ruijun Tian and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Nature Protocols感谢郑丽，田瑞军和其他匿名审稿人对这项工作的同行评审做出的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Related linksKey references using this protocolSojitra, M. et al. Nat. Chem. Biol. 17, 806–816 (2021): https://doi.org/10.1038/s41589-021-00788-5Lin, C.-L.

Additional informationPublisher的注释Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。。生物学17806-816（2021）：https://doi.org/10.1038/s41589-021-00788-5Lin，C.-L。

et al. Nat. Commun. 14, 5237 (2023): https://doi.org/10.1038/s41467-023-40900-ySchmidt, E. N. et al. Nat. Commun. 14, 2327 (2023): https://doi.org/10.1038/s41467-023-38030-6Extended dataExtended Data Fig. 1 Describes the workflow for generating the encoding glycan using SDB M13 phage.a) The M13KE SDB SVEK library is a combination of two degenerate codon regions in the phage DNA: SB1 and SB2.

。145237（2023年）：https://doi.org/10.1038/s41467-023-40900-ySchmidt，E.N.等人，《自然公社》。142327（2023年）：https://doi.org/10.1038/s41467-023-38030-6Extended数据扩展数据图1描述了使用SDB M13噬菌体产生编码聚糖的工作流程。a）M13KE SDB SVEK文库是噬菌体DNA中两个简并密码子区域的组合：SB1和SB2。

The combination of SB1 and SB2 yields a total of 1.3 × 1010 DNA possible barcoded phages that are phenotypically identical. b) The M13KE SDB SVEK library was plated at ~100 plaques per plate. Each plaque (clone) was isolated, individually amplified, and purified with Triton X-100 and PEG to remove lipopolysaccharides.

SB1和SB2的组合产生总共1.3×1010个表型相同的DNA可能的条形码噬菌体。b） M13KE SDB SVEK文库以每个板约100个斑块的速度铺板。分离每个斑块（克隆），单独扩增，并用Triton X-100和PEG纯化以去除脂多糖。

c) Schematic showing unmodified phage, DBCO-modified phage, and phage with azido-glycan. d) Workflow for modification of clonal phage with a distinct barcode. First, the phage is reacted with DBCO-NHS and verified by MALDI-TOF. Azide glycan is ligated with the DBCO on the phage. e) Typical MALDI-TOF spectra of unmodified phage, phage modified with DBCO, and after cycloaddition of azide glycan.Source dataExtended Data Fig.

c） 示意图显示未修饰的噬菌体，DBCO修饰的噬菌体和具有叠氮聚糖的噬菌体。d） 用不同条形码修饰克隆噬菌体的工作流程。首先，噬菌体与DBCO-NHS反应并通过MALDI-TOF验证。叠氮化物聚糖与噬菌体上的DBCO连接。e） 未修饰的噬菌体，用DBCO修饰的噬菌体以及叠氮化物聚糖环加成后的典型MALDI-TOF光谱。源数据扩展数据图。

2 Optimizing LiGA binding, washing, elution, and PCR.a) Incubation at 37 °C consistently showed higher amounts of eluted phage particles, n=3. b) Titer results describe the binding of a specific reporter phage decorated with DC-SIGN binding glycan (αMan-green) over multiple washes, n=3. Also shown are lactose (red), blank phage (white) and a po.

2优化LiGA结合，洗涤，洗脱和PCR。a）在37℃下孵育始终显示出更高量的洗脱噬菌体颗粒，n=3。b） 滴度结果描述了用DC-SIGN结合聚糖（αMan green）修饰的特异性报告噬菌体在多次洗涤中的结合，n=3。还显示了乳糖（红色），空白噬菌体（白色）和po。

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