Departments of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA.
Precision Vaccines Program, Boston Children's Hospital, Boston, MA, USA.
Adv Exp Med Biol. 2021;1306:1-12. doi: 10.1007/978-3-030-63908-2_1.
Proteomics analysis of urine samples allows for studying the impact of system perturbation. However, meaningful proteomics-based biomarker discovery projects often require the analysis of large patient cohorts with hundreds of samples to describe the biological variability. Thus, robust high-throughput sample processing methods are a prerequisite for clinical proteomics pipelines that minimize experimental bias due to individual sample processing methods. Herein we describe a high-throughput method for parallel 96-well plate-based processing of urine samples for subsequent LC/MS-based proteomic analyses. Protein digestion and subsequent sample processing steps are efficiently performed in 96-well polyvinylidene fluoride (PVDF) membrane plate allowing for the use of vacuum manifolds for rapid liquid transfer, and multichannel pipettes and/or liquid handing robots. In this chapter we make available a detailed step-by-step protocol for our 'MStern blotting' sample processing strategy applied to patient urine samples followed by mass spectrometry-based proteomics analysis. Subsequently, we provide an example application using minimal volume of urine samples (e.g. 150 μL) collected from children pre and post thoracotomy to identify the predominant sites of protein catabolism and aid in the design of therapies to ameliorate protein catabolism and breakdown during critical illness. Furthermore, we demonstrate how the systemic state is reflected in the urine as an easily obtainable, stable, and safe biofluid.
尿液样本的蛋白质组学分析可用于研究系统干扰的影响。然而，有意义的基于蛋白质组学的生物标志物发现项目通常需要对数百个样本的大患者队列进行分析，以描述生物学变异性。因此，稳健的高通量样本处理方法是临床蛋白质组学管道的前提，可最大限度地减少由于个别样本处理方法引起的实验偏差。在此，我们描述了一种高通量的方法，用于并行处理 96 孔板的尿液样本，以便进行随后的基于 LC/MS 的蛋白质组学分析。蛋白质消化和随后的样品处理步骤在 96 孔聚偏二氟乙烯（PVDF）膜板中高效进行，允许使用真空歧管进行快速液体转移，以及多通道移液器和/或液体处理机器人。在这一章中，我们提供了一个详细的分步协议，用于我们的“MStern 印迹”样品处理策略，应用于患者尿液样本，随后进行基于质谱的蛋白质组学分析。随后，我们提供了一个使用最小体积尿液样本（例如，150μL）的示例应用，这些样本取自胸腔镜手术前后的儿童，以确定蛋白质分解的主要部位，并有助于设计治疗方案以改善危重病期间的蛋白质分解和分解。此外，我们展示了系统状态如何反映在尿液中，作为一种易于获得、稳定和安全的生物流体。
