The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD, 20850, USA,
Adv Exp Med Biol. 2015;845:77-87. doi: 10.1007/978-94-017-9523-4_8.
Urine has been an important body fluid source for diagnostic and prognostic biomarkers of diseases for a long time. Technological advances during the last two decades have enabled a fundamental shift from the discovery of candidate protein biomarkers using single-assay platforms to highly parallel liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomic analysis platforms. MS/MS-based approaches such as multiple reaction monitoring (MRM) are also being used increasingly for targeted protein biomarker validation. In large part due to the fact that the majority of protein in voided urine is soluble, such studies have focused on the analysis of urine supernatants, whereas the pellets were discarded after centrifugal sedimentation. Urine sediments are of particular value in the analysis of urinary tract infections (UTI). The LC-MS/MS methods now have sufficient resolving power and sensitivity to survey metaproteomes--the entirety of proteins derived from multiple organisms that interact with each other in mutualistic or antagonistic fashion. Challenges of proteomic analysis of urine include the high dynamic range of protein abundance, high levels of protein post-translational modifications, and high quantities of natural protease inhibitors. Recently, a robust and scalable workflow that can parallelize the processing of multiple urinary supernatant and sediment samples was developed and validated in our lab. This method utilizes 96-well format filter-aided sample preparation (96FASP) strategy and was shown to successfully identify large numbers of proteins from urine samples. Processing 10-50 µg total protein in single experiment, LC-MS/MS with a Q-Exactive mass spectrometer resulted in more than 1,100 distinct human protein identifications from urine supernatants, and around 400 microbial and 1,400 human protein identifications from urine sediments. The surveys are a rich data resource not only for biomarker discovery but also to interrogate mechanisms of pathogenesis in the urinary system.
尿液一直是疾病诊断和预后生物标志物的重要体液来源。在过去的二十年中，技术的进步使得从使用单一分析平台发现候选蛋白质生物标志物到基于高平行液相色谱串联质谱（LC-MS/MS）的蛋白质组分析平台的根本性转变成为可能。基于 MS/MS 的方法，如多重反应监测（MRM），也越来越多地用于靶向蛋白质生物标志物验证。在很大程度上是由于尿液中大多数蛋白质都是可溶性的，因此这些研究主要集中在尿液上清液的分析上，而离心沉淀后则丢弃沉淀物。尿液沉淀物在尿路感染（UTI）分析中具有特别的价值。LC-MS/MS 方法现在具有足够的分辨率和灵敏度来调查元蛋白质组——来自相互以互利或拮抗方式相互作用的多个生物体的全部蛋白质。尿液蛋白质组分析的挑战包括蛋白质丰度的高动态范围、蛋白质翻译后修饰水平高和天然蛋白酶抑制剂数量多。最近，我们实验室开发并验证了一种强大且可扩展的工作流程，可以并行处理多个尿液上清液和沉淀物样本。该方法利用 96 孔格式过滤辅助样品制备（96FASP）策略，成功地从尿液样本中鉴定出大量蛋白质。在单个实验中处理 10-50 µg 总蛋白，使用 Q-Exactive 质谱仪的 LC-MS/MS 可从尿液上清液中鉴定出超过 1,100 种不同的人类蛋白质，从尿液沉淀物中鉴定出约 400 种微生物和 1,400 种人类蛋白质。这些调查不仅是生物标志物发现的丰富数据资源，也是研究泌尿系统发病机制的重要手段。
