PDF(Pubmed)
Abstract：
Urine provides a diverse source of information related to a patient\'s health status and is ideal for clinical proteomics due to its ease of collection. To date, most methods for the preparation of urine samples lack the throughput required to analyze large clinical cohorts. To this end, we developed a novel workflow, urine-HILIC (uHLC), based on an on-bead protein capture, clean-up, and digestion without the need for bottleneck processing steps such as protein precipitation or centrifugation. The workflow was applied to an acute kidney injury (AKI) pilot study. Urine from clinical samples and a pooled sample was subjected to automated sample preparation in a KingFisher™ Flex magnetic handling station using the novel approach based on MagReSyn® HILIC microspheres. For benchmarking, the pooled sample was also prepared using a published protocol based on an on-membrane (OM) protein capture and digestion workflow. Peptides were analyzed by LCMS in data-independent acquisition (DIA) mode using a Dionex Ultimate 3000 UPLC coupled to a Sciex 5600 mass spectrometer. The data were searched in Spectronaut™ 17. Both workflows showed similar peptide and protein identifications in the pooled sample. The uHLC workflow was easier to set up and complete, having less hands-on time than the OM method, with fewer manual processing steps. Lower peptide and protein coefficient of variation was observed in the uHLC technical replicates. Following statistical analysis, candidate protein markers were filtered, at ≥8.35-fold change in abundance, ≥2 unique peptides and ≤1% false discovery rate, and revealed 121 significant, differentially abundant proteins, some of which have known associations with kidney injury. The pilot data derived using this novel workflow provide information on the urinary proteome of patients with AKI. Further exploration in a larger cohort using this novel high-throughput method is warranted.
摘要：
尿液提供了与患者健康状况相关的多种信息来源，并且由于其易于收集而成为临床蛋白质组学的理想选择。迄今为止,大多数制备尿样的方法缺乏分析大型临床队列所需的通量.为此,我们开发了一个新的工作流程，尿HILIC（uHLC），基于珠子上的蛋白质捕获，清理,和消化，而不需要瓶颈处理步骤，如蛋白沉淀或离心。该工作流程应用于急性肾损伤(AKI)初步研究。使用基于MagReSyn®HILIC微球的新颖方法,在KingFisher™Flex磁性处理站中对来自临床样品和合并样品的尿液进行自动化样品制备。对于基准测试，合并样品也是使用基于膜上(OM)蛋白质捕获和消化工作流程的公开方案制备的.使用与Sciex5600质谱仪偶联的DionexUltimate3000UPLC,通过LCMS以数据无关采集(DIA)模式分析肽。在Spectronaut™17中搜索数据。两个工作流程在合并的样品中显示相似的肽和蛋白质鉴定。uHLC工作流程更容易设置和完成，动手时间比OM方法少，更少的手动处理步骤。在uHLC技术重复中观察到较低的肽和蛋白质变异系数。经过统计分析，候选蛋白质标记物被过滤，在丰度变化≥8.35倍时，≥2种独特肽和≤1%的错误发现率，并揭示了121个重要的，差异丰富的蛋白质，其中一些已知与肾损伤有关。使用这种新颖的工作流程获得的试验数据提供了有关AKI患者的尿蛋白质组的信息。使用这种新颖的高通量方法在更大的群体中进行进一步的探索是有必要的。
