Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research.
Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research.
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DOI：
10.1038/s41598-017-02599-y
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发表时间：
2017-06-02
影响因子：
4.6
通讯作者：
Hendrix A
中科院分区：
文献类型：
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作者：
Vergauwen G;Dhondt B;Van Deun J;De Smedt E;Berx G;Timmerman E;Gevaert K;Miinalainen I;Cocquyt V;Braems G;Van den Broecke R;Denys H;De Wever O;Hendrix A
细胞外囊泡（EV）相关生物标志物的鉴定和验证需要稳健的分离和表征方案。我们评估了EV研究中一些常用的分析前、分析后和分析后变量的影响。具有不同膜类型和孔径的离心过滤器用于在EV分离之前减少大体积生物流体或浓缩EV。我们比较了五种常用的过滤器在使用血浆、尿液和EV加标PBS时的效率。孔径为10 kDa的再生纤维素膜回收EV的效率最高。其他过滤器的回收率低于40%。接下来，我们分析了蛋白质测定类型对在比色和荧光试剂盒中测量EV蛋白质的影响。荧光测定Qubit测量低浓度EV和BSA样品最准确，技术和生物重复之间的变化最小。最后，我们定量了来自密度梯度超离心的EV样品中的Optiprep残留物，并证明了尺寸排阻色谱法可有效地从EV中去除Optiprep。总之，离心过滤器和蛋白质测定的选择混淆了EV分析，应仔细考虑以提高生物标志物发现的效率。下游应用可以考虑基于SEC从EV中去除Optiprep残留物。
Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.