Toward Biomarker Development in Large Clinical Cohorts: An Integrated High-Throughput 96-Well-Plate-Based Sample Preparation Workflow for Versatile Downstream Proteomic Analyses
Toward Biomarker Development in Large Clinical Cohorts: An Integrated High-Throughput 96-Well-Plate-Based Sample Preparation Workflow for Versatile Downstream Proteomic Analyses
复制标题
面向大型临床队列的生物标志物开发：基于 96 孔板的集成高通量样品制备工作流程，用于多功能下游蛋白质组分析
DOI：
10.1021/acs.analchem.6b01333
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发表时间：
2016
影响因子：
7.4
通讯作者：
Li Lanjuan
中科院分区：
文献类型：
--
作者：
Sun Zeyu;Liu Xiaoli;Jiang Jing;Huang Haijun;Wang Jie;Wu Daxian;Li Lanjuan
我们描述了一种廉价、稳健、快速、高通量和灵活的蛋白质组学样品处理方法，该方法基于常规的96孔板，在低离心速度下通过丙酮沉淀（96 PACS），该方法能够在2 h内对96个样品进行预消化处理。在复杂的Huh-7总裂解物上进行测试，96 PACS产生了可比的蛋白质组覆盖率，甚至显示出比FASP更好的再现性。使用数据独立采集和平行反应监测定量在一组6个基准样品中进一步测试96 PACS的定量性能，所述基准样品由在复合物E中加标的BSA的6个系列稀释液组成。大肠杆菌蛋白质组背景。该方案还成功地修改了自动化，并在比较无标记蛋白质组学研究中得到验证，以开发血清标志物，用于早期检测慢性B型肝炎病毒感染患者的肝纤维化和坏死性炎症。
We describe a cheap, robust, fast, high-throughput, and flexible proteomic sample processing method based on a regular 96-well plate by acetone precipitation under low centrifuge speed (96PACS), which enables predigestion processing of 96 samples within 2 h. Tested on a complex Huh-7 total lysate, 96PACS produced comparable proteome coverage and even showed better reproducibility than FASP. Quantitative performance of 96PACS was further tested using data-independent acquisition and parallel reaction monitoring quantitation in a set of 6 benchmark samples consisting of 6 serial dilutions of BSA spiked in complexE. coliproteome background. The protocol was also successfully modified for automation and was validated in a comparative label-free proteomic study to develop serum markers for early detection of liver fibrosis and necroinflammation in patients chronically infected with hepatitis B virus.