Mass spectrometry for highly sensitive and sample-sparing analysis of extracellular vesicles in liver diseases

摘要 非酒精性脂肪性肝病(NAFLD)是美国最常见的肝病。NAFLD是一种 相对非进行性非酒精性脂肪肝(NAFL)和进展性非酒精性脂肪肝的聚集性 脂肪肝(NASH)。虽然肝脏脂肪变性是NAFL和NASH的保守特征，但后者是 以肝损伤、炎症、纤维化以及患肝癌和肝硬变的风险为特征。目前， 没有有效的非侵入性和可扩展的筛查策略来区分NAFL和NASH 也不监控纳什的进展。因此，对相对容易的生物标记物存在着巨大的未得到满足的需求 为了获得，可以随着时间的推移反复检测，可以区分NAFL和NASH，在病理生理上是 形成，并帮助对NASH患者进行风险分层。细胞外小泡(EV)携带来自患病细胞的信号 和器官，因此是NAFL和NASH的理想生物标志物。然而，电动汽车的发展 基于生物标记物的研究受到了几个挑战的困扰。第一个挑战与大样本有关 传统上用于电动汽车分析的质谱学方法的体积要求(例如LC-MS/MS)。这 限制了它们对稀缺患者样本分析的实用性。第二个挑战在于电动汽车的发布 从患病的肝脏通过循环稀释，只占总EVS的一小部分(~1%) 存在于外周血中。这使得识别疾病进展或治疗复发的生物标志物变得困难。 海绵。在这个项目中，我们将通过开发新技术来应对上述挑战：1) 纳米弹丸(NP)二次离子质谱仪(SIMS)，将实现对电动汽车的多路分析，使用 微升样本和2)微流控器官型肝脏培养，使我们能够采集未稀释的肝脏EV 来自健康或患病的组织。 该项目完成后，我们将拥有强大的蛋白质组学质谱学技术。 和EVS的脂肪组学分析。在这个项目中，电动汽车生物标志物的识别将在未来进行。 以血液为基础的测试，将允许诊断NAFLD，区分NAFL和NASH，并帮助风险分层 病人。这样的检测将取代目前用于诊断的高侵入性肝活检，并将带来革命性的变化 非酒精性脂肪肝患者的护理。它还将允许在高危人群中早期诊断NASH。

ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease in the United States. NAFLD is a conglomerate of the relatively nonprogressive non-alcoholic fatty liver (NAFL) and progressive non-alcoholic steatohepatitis (NASH). While hepatic steatosis is a conserved feature of both NAFL and NASH, the latter is characterized by liver injury, inflammation, fibrosis, and the risk of liver cancer and cirrhosis. At the present time, there are no effective noninvasive and scalable screening strategies to distinguish between NAFL and NASH nor monitor NASH progression. Thus, there is a significant unmet need for biomarkers which are relatively easy to obtain, can be tested repeatedly over time, can distinguish NAFL from NASH, are pathophysiologically in- formed, and help risk stratify patients with NASH. Extracellular vesicles (EVs) carry signals from diseased cells and organs and therefore represent ideal biomarkers for NAFL and NASH. However, the development of EV based biomarkers has been confounded by several challenges. First challenge relates to the large sample volume requirements of mass spectrometry approaches traditionally used for EV analysis (e.g. LC-MS/MS). This limits their utility for the analysis of scarce patient samples. Second challenge lies in the fact that EVs released from the diseased liver become diluted by circulation and represent only a small subset (~1%) of total EVs present in peripheral blood. This makes it difficult to identify biomarkers of disease progression or therapy re- sponse. In this project, we will address the aforementioned challenges by developing novel technologies: 1) nanoprojectile (NP) secondary ion mass spectrometry (SIMS) that will enable multiplexed analysis of EVs using microliters of sample and 2) microfluidic organotypic liver cultures that will allow us to harvest undiluted liver EVs from healthy or diseased tissue. Upon completion of this project, we will have in hand a powerful mass spectrometry technology for proteomic and lipidomic analysis of EVs. The identification of EV biomarkers in this project will, in the future, be parlayed into a blood-based test that will allow to diagnose NAFLD, distinguish NAFL from NASH and help risk stratify patients. Such a test will replace highly invasive liver biopsy currently used for diagnosis and will revolutionize the care of patients with NAFLD. It will also allow early diagnosis of NASH in at risk populations.