Which casein micelle removal method is suitable for studies of human milk extracellular vesicles? A systematic comparison of four different treatments for casein depletion before extracellular vesicle isolation from human milk

Hatice Cetinkaya, Supasek Kongsomros, Laurie Nommsen-Rivers, Ardythe L. Morrow, S. Chutipongtanate
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Abstract

Aim: This study aimed to systematically compare four casein micelle removal methods on the particle and protein characteristics of the isolated human milk EVs. Methods: The defatted milk was treated with 1% sodium citrate, 20 mM ethylenediaminetetraacetic acid (EDTA), 1% acetic acid, or 1% chymosin/calcium chloride for 30 min at 4 °C to remove casein micelles. EV isolation was performed using qEV size exclusion chromatography. Milk turbidity at the optical density 350 nm and dot immunoblot with casein antibody were applied to monitor the qEV fractions. Particle analyses were performed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The enrichment of human milk EV markers, i.e., tetraspanins, Alix, lactadherin, butyrophilin, and xanthine dehydrogenase, and casein depletion capabilities were evaluated by proteomics and immunoblotting. Results: Compared to the untreated condition, sodium citrate and EDTA decreased milk turbidity by disrupting casein micelles, while acetic acid and chymosin removed them by inducing precipitation/coagulation. All treatments shifted casein immunoreactivity in the qEV fractions from large micelles (the exclusion volume) to small molecular sizes (gel-infiltrated fractions). Acidification affected human milk EV morphology, while EDTA, acetic acid, and chymosin methods slightly altered EV particle numbers. Different casein micelle removal methods confer different degrees of human milk EV marker enrichment and casein depletion. The method performances could be ranked as follows: chymosin > EDTA > acetic acid > sodium citrate. Conclusion: Our findings suggest that chymosin and EDTA should be considered as the method of choice for casein micelle removal in future studies involving human milk EV isolation and characterization.
哪种酪蛋白胶束去除方法适合人乳细胞外囊泡研究?系统比较从母乳中分离胞外囊泡前的四种不同酪蛋白去除处理方法
目的:本研究旨在系统比较四种酪蛋白胶束去除方法对分离出的人乳EVs的颗粒和蛋白质特征的影响。方法:将脱脂牛奶用 1%柠檬酸钠、20 mM 乙二胺四乙酸处理:脱脂牛奶经1%柠檬酸钠、20 mM乙二胺四乙酸(EDTA)、1%醋酸或1%糜蛋白酶/氯化钙在4 ℃下处理30分钟,以去除酪蛋白胶束。使用 qEV 排阻色谱法分离 EV。用光密度为 350 nm 的牛奶浑浊度和酪蛋白抗体点免疫印迹来监测 qEV 产物。利用透射电子显微镜(TEM)和纳米粒子跟踪分析(NTA)对粒子进行分析。通过蛋白质组学和免疫印迹法评估了人乳 EV 标记(即四泛蛋白、Alix、乳粘连蛋白、丁淀粉蛋白和黄嘌呤脱氢酶)的富集和酪蛋白耗竭能力。结果与未处理的情况相比,柠檬酸钠和乙二胺四乙酸通过破坏酪蛋白胶束来降低牛奶的浑浊度,而醋酸和糜蛋白酶则通过诱导沉淀/凝固来去除酪蛋白胶束。所有处理方法都会使qEV馏分中的酪蛋白免疫反应从大胶束(排除体积)转移到小分子大小(凝胶渗透馏分)。酸化会影响人乳EV的形态,而乙二胺四乙酸、醋酸和糜蛋白酶方法会轻微改变EV颗粒的数量。不同的酪蛋白胶束去除方法对人乳 EV 标记富集和酪蛋白耗竭的程度不同。不同方法的性能排名如下:糜蛋白酶 > EDTA > 乙酸 > 柠檬酸钠。结论我们的研究结果表明,在今后涉及人乳 EV 分离和表征的研究中,糜蛋白酶和 EDTA 应被视为去除酪蛋白胶束的首选方法。
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