Surface Sialic Acid Detection of Small Extracellular Vesicles at the Single-Particle Level by Nano-Flow Cytometry,Analytical Chemistry

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Surface Sialic Acid Detection of Small Extracellular Vesicles at the Single-Particle Level by Nano-Flow Cytometry
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-07-24 , DOI: 10.1021/acs.analchem.4c01763
Niangui Cai ₁ , Xiaozhen Zhan ₁ , Yan Chen ₁ , Junwei Xue ₁ , Chen Chen ₁ , Yurou Li ₁ , Ye Tian ₁ , Xiaomei Yan ₁

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Glycans, particularly sialic acids (SAs), play crucial roles in diverse biological processes. Despite their significance, analyzing specific glycans, such as sialic acids, on individual small extracellular vesicles (sEVs) has remained challenging due to the limited glycan capacity and substantial heterogeneity of sEVs. To tackle this issue, we introduce a chemical modification method of surface SAs on sEVs named PALEV-nFCM, which involves periodate oxidation and aniline-catalyzed oxime ligation (PAL), in conjunction with single-particle analysis using a laboratory-built nano-flow cytometer (nFCM). The specificity of the PALEV labeling method was validated using SA-decorated liposomes, enzymatic removal of terminal SA residues, lectin preblocking, and cellular treatment with an endogenous sialyltransferase inhibitor. Comprehensive mapping of SA distributions was conducted for sEVs derived from different sources, including conditioned cell culture medium (CCCM) of various cell lines, human saliva, and human red blood cells (RBCs). Notably, treatment with the calcium ionophore substantially increases the population of SA-positive RBC sEVs and enhances the SA content on individual RBC sEVs as well. nFCM provides a sensitive and versatile platform for mapping SAs of individual sEVs, which could significantly contribute to resolving the heterogeneity of sEVs and advancing the understanding of their glycosignature.

中文翻译：

通过纳米流式细胞术在单颗粒水平检测小细胞外囊泡的表面唾液酸

聚糖，特别是唾液酸 (SA)，在多种生物过程中发挥着至关重要的作用。尽管其意义重大，但由于 sEV 的聚糖容量有限和显着的异质性，分析单个小细胞外囊泡 (sEV) 上的特定聚糖（例如唾液酸）仍然具有挑战性。为了解决这个问题，我们引入了一种名为 PALEV-nFCM 的 sEV 表面 SA 化学修饰方法，该方法涉及高碘酸盐氧化和苯胺催化肟连接（PAL），并结合使用实验室构建的纳米流进行单颗粒分析细胞计数仪（nFCM）。 PALEV 标记方法的特异性使用 SA 修饰的脂质体、末端 SA 残基的酶促去除、凝集素预封闭以及内源性唾液酸转移酶抑制剂的细胞处理进行了验证。对不同来源的 sEV 进行了 SA 分布的全面绘图，包括各种细胞系的条件细胞培养基 (CCCM)、人类唾液和人类红细胞 (RBC)。值得注意的是，钙离子载体治疗显着增加了 SA 阳性红细胞 sEV 的数量，并提高了单个红细胞 sEV 上的 SA 含量。 nFCM 为绘制单个 sEV 的 SA 提供了一个灵敏且多功能的平台，这可以极大地有助于解决 sEV 的异质性并促进对其糖签名的理解。

更新日期：2024-07-24

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