Royal Institute of Technology (KTH), School of Engineering Sciences in Chemistry, Biotechnology and Health, Department of Gene Technology, Science for Life Laboratory, Solna, Sweden.
Royal Institute of Technology (KTH), School of Engineering Sciences in Chemistry, Biotechnology and Health, Department of Protein Science, AlbaNova University Center, Stockholm, Sweden.
J Extracell Vesicles. 2022 Nov;11(11):e12277. doi: 10.1002/jev2.12277.
Small extracellular vesicles (sEVs) have in recent years evolved as a source of biomarkers for disease diagnosis and therapeutic follow up. sEV samples derived from multicellular organisms exhibit a high heterogeneous repertoire of vesicles which current methods based on ensemble measurements cannot capture. In this work we present droplet barcode sequencing for protein analysis (DBS-Pro) to profile surface proteins on individual sEVs, facilitating identification of sEV-subtypes within and between samples. The method allows for analysis of multiple proteins through use of DNA barcoded affinity reagents and sequencing as readout. High throughput single vesicle profiling is enabled through compartmentalization of individual sEVs in emulsion droplets followed by droplet barcoding through PCR. In this proof-of-concept study we demonstrate that DBS-Pro allows for analysis of single sEVs, with a mixing rate below 2%. A total of over 120,000 individual sEVs obtained from a NSCLC cell line and from malignant pleural effusion (MPE) fluid of NSCLC patients have been analyzed based on their surface proteins. We also show that the method enables single vesicle surface protein profiling and by extension characterization of sEV-subtypes, which is essential to identify the cellular origin of vesicles in heterogenous samples.
近年来，小细胞外囊泡 (sEVs) 已成为疾病诊断和治疗随访的生物标志物来源。从多细胞生物中提取的 sEV 样本表现出高度异质的囊泡谱，而目前基于整体测量的方法无法捕获这些囊泡。在这项工作中，我们提出了用于蛋白质分析的液滴条码测序 (DBS-Pro)，以对单个 sEV 上的表面蛋白进行分析，从而促进了对样本内和样本间 sEV 亚型的鉴定。该方法通过使用 DNA 条码亲和试剂和测序作为读出，允许分析多种蛋白质。通过将单个 sEV 分隔在乳液液滴中，并通过 PCR 进行液滴条码化，实现了高通量的单个囊泡分析。在这项概念验证研究中，我们证明了 DBS-Pro 允许分析单个 sEV，混合率低于 2%。总共分析了来自 NSCLC 细胞系和 NSCLC 患者恶性胸腔积液 (MPE) 液体的超过 120,000 个单个 sEV，其依据是它们的表面蛋白。我们还表明，该方法能够对单个囊泡表面蛋白进行分析，从而扩展了 sEV 亚型的特征描述，这对于识别异质样本中囊泡的细胞来源至关重要。
