Laboratory of Clinical Omics Research, Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan.
Institute for Human Life Innovatiaon, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan.
J Proteome Res. 2022 Sep 2;21(9):2085-2093. doi: 10.1021/acs.jproteome.2c00121. Epub 2022 Aug 1.
Proteomics has become an increasingly important tool in medical and medicinal applications. It is necessary to improve the analytical throughput for these applications, particularly in large-scale drug screening to enable measurement of a large number of samples. In this study, we aimed to establish an ultrafast proteomic method based on 5-min gradient LC and quadrupole-Orbitrap mass spectrometer (Q-Orbitrap MS). We precisely optimized data-independent acquisition (DIA) parameters for 5-min gradient LC and reached a depth of >5000 and 4200 proteins from 1000 and 31.25 ng of HEK293T cell digest in a single-shot run, respectively. The throughput of our method enabled the measurement of approximately 80 samples/day, including sample loading, column equilibration, and wash running time. We demonstrated that our method is applicable for the screening of chemical responsivity via a cell stimulation assay. These data show that our method enables the capture of biological alterations in proteomic profiles with high sensitivity, suggesting the possibility of large-scale screening of chemical responsivity.
蛋白质组学已成为医学和药物应用中越来越重要的工具。对于这些应用，特别是在大规模药物筛选中，需要提高分析通量，以实现对大量样本的测量。在本研究中，我们旨在建立一种基于 5 分钟梯度 LC 和四极杆-Orbitrap 质谱仪（Q-Orbitrap MS）的超快蛋白质组学方法。我们精确优化了 5 分钟梯度 LC 的数据非依赖性采集（DIA）参数，单次运行中分别从 1000 和 31.25ng 的 HEK293T 细胞消化物中获得了 >5000 和 4200 种蛋白质。我们方法的通量可实现约 80 个样本/天的测量，包括样品加载、柱平衡和冲洗运行时间。我们证明了我们的方法适用于通过细胞刺激测定筛选化学反应性。这些数据表明，我们的方法能够以高灵敏度捕获蛋白质组图谱中的生物学变化，提示有可能进行大规模的化学反应性筛选。
