Lin Lin, Yu Quan, Zheng Jiaxin, Cai Zonglong, Tian Ruijun
Materials Characterization and Preparation Center, Southern University of Science and Technology, Shenzhen, 518055 China.
2Division of Advanced Manufacturing, Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055 China.
Clin Proteomics. 2018 Dec 22;15:42. doi: 10.1186/s12014-018-9220-2. eCollection 2018.
Urine has evolved as a promising body fluids in clinical proteomics because it can be easily and noninvasively obtained and can reflect physiological and pathological status of the human body. Many efforts have been made to characterize more urinary proteins in recent years, but few have focused on the analysis throughput and detection reproducibility. Increasing the urine proteomic profiling throughput and reproducibility is urgently needed for discovering potential biomarker in large cohorts.
In this study, we developed a fast and robust workflow for streamlined urinary proteome analysis. The workflow integrate highly efficient sample preparation technique and urinary specific data-independent acquisition (DIA) approach. The performance of the workflow was systematically evaluated and the workflow was subsequently applied in a proof-of-concept urine proteome study of 21 kidney cancer (KC) patients and 22 healthy controls.
With this workflow, the entire sample preparation process takes less than 3 h and allows multiplexing on standard centrifuges. Without pre-fractionation, our newly developed DIA method allows quantitative analysis of ~ 1000 proteins within 80 min of MS time (~ 15 samples/day). The quantitation accuracy of the whole workflow was excellent with median CV of 9.1%. The preliminary study on KC identified 125 significantly changed proteins.
The result suggested the feasibility of applying the high throughput workflow in extensive urinary proteome profiling and clinical relevant biomarker discovery.
尿液已成为临床蛋白质组学中一种很有前景的体液，因为它可以轻松、无创地获取，并且能够反映人体的生理和病理状态。近年来，人们为鉴定更多的尿液蛋白质付出了诸多努力，但很少有人关注分析通量和检测重现性。在大规模队列中发现潜在生物标志物迫切需要提高尿液蛋白质组分析的通量和重现性。
在本研究中，我们开发了一种用于简化尿液蛋白质组分析的快速且稳健的工作流程。该工作流程整合了高效的样品制备技术和尿液特异性的数据非依赖采集（DIA）方法。我们系统地评估了该工作流程的性能，并随后将其应用于一项针对21例肾癌（KC）患者和22例健康对照的概念验证尿液蛋白质组研究中。
通过此工作流程，整个样品制备过程耗时不到3小时，并且可以在标准离心机上进行多重操作。在不进行预分级分离的情况下，我们新开发的DIA方法能够在80分钟的质谱分析时间内（约每天15个样品）对约1000种蛋白质进行定量分析。整个工作流程的定量准确性极佳，中位变异系数为9.1%。对肾癌的初步研究鉴定出125种显著变化的蛋白质。
结果表明将高通量工作流程应用于广泛的尿液蛋白质组分析和临床相关生物标志物发现具有可行性。
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