Proteomics Core Facility, Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, Pennsylvania 19104, United States.
Division of Urology, Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, Pennsylvania 19104, United States.
J Proteome Res. 2020 Apr 3;19(4):1857-1862. doi: 10.1021/acs.jproteome.9b00772. Epub 2020 Mar 10.
The growing field of urinary proteomics shows promise to expand the number of biomarkers for the diagnosis and prognosis of a number of human diseases. With the rapid developments in mass spectrometry methods for proteome quantification, there exists an opportunity for improved sample processing and separation workflows to make important contributions to urine proteomic analyses. Here we evaluate the performance of four sample preparation methods: MStern, PreOmics in-StageTip (iST), suspension-trapping (S-Trap), and conventional urea In-Solution trypsin hydrolysis for nondepleted urine samples. Data-dependent acquisition (DDA) mode on a QExactive HF mass spectrometer was used for single-shot label-free data acquisition. Our results demonstrate a high degree of reproducibility within each workflow. PreOmics iST yields the best digestion efficiency, whereas the S-Trap workflow gives the greatest number of peptide and protein identifications. Using the S-Trap method and starting with ∼0.5 mL, we identify ∼1500 protein groups and ∼17 700 peptides from DDA analysis with a single injection on the mass spectrometer.
尿液蛋白质组学领域的发展前景广阔，有望增加许多人类疾病的诊断和预后的生物标志物数量。随着用于蛋白质组定量的质谱方法的快速发展，有机会改进样品处理和分离工作流程，为尿液蛋白质组分析做出重要贡献。在这里，我们评估了四种样品制备方法的性能：MStern、PreOmics in-StageTip (iST)、悬浮捕获 (S-Trap) 和常规尿素 In-Solution 胰蛋白酶水解法用于非耗竭尿液样品。在 QExactive HF 质谱仪上使用数据依赖型采集 (DDA) 模式进行单次无标记数据采集。我们的结果表明每个工作流程内具有高度的重现性。PreOmics iST 具有最佳的消化效率，而 S-Trap 工作流程则提供了最多的肽和蛋白质鉴定。使用 S-Trap 方法，从约 0.5 mL 开始，我们通过在质谱仪上单次注射，从 DDA 分析中鉴定出约 1500 个蛋白质组和约 17700 个肽。
