Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States.
Anal Chem. 2022 May 24;94(20):7164-7168. doi: 10.1021/acs.analchem.2c01252. Epub 2022 May 11.
Exosomes are small extracellular vesicles (EVs) secreted by all cells and found in biological fluids, which can serve as minimally invasive liquid biopsies with extremely high therapeutic and diagnostic potential. Mass spectrometry (MS)-based proteomics is a powerful technique to profile and quantify the protein content in exosomes, but the current methods require laborious and time-consuming multistep sample preparation that significantly limit throughput. Herein, we report a one-pot exosome proteomics method enabled by a photocleavable surfactant, Azo, to simplify exosomal lysis, effectively extract proteins, and expedite digestion. We have applied this method to exosomes derived from isolated mammary fibroblasts and confidently identified 3466 proteins and quantified 2288 proteins using a reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight mass spectrometer. Here, 3166 (91%) of the identified proteins are annotated in the exosome/EVs databases, ExoCarta and Vesiclepedia, including important exosomal markers, CD63, PDCD6IP, and SDCBP. This method is fast, simple, and highly effective at extracting exosomal proteins with high reproducibility for deep exosomal proteome coverage. We envision that this method could be generally applicable for exosome proteomics applications in biomedical research, therapeutic interventions, and clinical diagnostics.
外泌体是所有细胞分泌的小型细胞外囊泡 (EV)，存在于生物体液中，可作为具有极高治疗和诊断潜力的微创液体活检。基于质谱 (MS) 的蛋白质组学是一种对外泌体中的蛋白质含量进行分析和定量的强大技术，但目前的方法需要繁琐且耗时的多步样品制备，这极大地限制了通量。在此，我们报告了一种由光裂解表面活性剂 Azo 实现的一管式外泌体蛋白质组学方法，该方法简化了外泌体的裂解，有效地提取了蛋白质，并加快了消化速度。我们已经将这种方法应用于从分离的乳腺成纤维细胞中提取的外泌体，并使用反相液相色谱与被困离子淌度谱 (TIMS) 四极飞行时间质谱联用，自信地鉴定了 3466 种蛋白质，并定量了 2288 种蛋白质。在此，3166 种（91%）鉴定的蛋白质在 ExoCarta 和 Vesiclepedia 等外泌体/EVs 数据库中进行了注释，包括重要的外泌体标记物 CD63、PDCD6IP 和 SDCBP。该方法快速、简单，在外泌体蛋白质提取方面非常有效，具有高重复性，可实现深度外泌体蛋白质组覆盖。我们设想这种方法可以广泛应用于生物医学研究、治疗干预和临床诊断中的外泌体蛋白质组学应用。
