Spanish National Research Council (CSIC), 36208, Vigo, Spain.
Complutense University of Madrid (UCM) , 28040, Madrid, Spain.
Anal Chem. 2017 Sep 5;89(17):8853-8862. doi: 10.1021/acs.analchem.7b01321. Epub 2017 Aug 22.
We propose a new workflow for fast phosphoproteome profiling. The workflow is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) combined with an inverse strategy based on TiO selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX) and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a high-resolution mass spectrometer. The performance of the method was established for the global phosphoproteome analysis of unstimulated human Jurkat leukemia T cells (E6.1). Using this accelerated workflow, 15367 phosphorylation sites from 13029 different phosphopeptides belonging to 3163 different phosphoproteins were efficiently identified with high-throughput and reproducibility in less than 15 h. The functional analysis revealed significant phosphorylation-based networks that are implicated in immune function and tumor development pathways. The present strategy, HIFU-TiO-SCX-LC-MS/MS, is the fastest analytical method reported to date for generating large-scale phosphoproteomics data sets (<15 h).
我们提出了一种新的快速磷酸肽组学分析工作流程。该工作流程基于在高强度聚焦超声（HIFU）提供的超声场下加速溶液内胰蛋白酶消化，结合基于 TiO2 的反向策略选择性磷酸肽富集、强阳离子交换色谱（SCX）分级和使用高分辨率质谱仪的液相色谱串联质谱（LC-MS/MS）分析。该方法的性能已通过未刺激的人 Jurkat 白血病 T 细胞（E6.1）的全磷酸肽组分析得到确立。使用这种加速工作流程，在不到 15 小时的时间内，高效地鉴定了来自 3163 种不同磷酸化蛋白的 13029 种不同磷酸肽中的 15367 个磷酸化位点，具有高通量和重现性。功能分析揭示了涉及免疫功能和肿瘤发展途径的基于显著磷酸化的网络。目前的策略 HIFU-TiO2-SCX-LC-MS/MS 是迄今为止报道的用于生成大规模磷酸肽组学数据集（<15 小时）的最快分析方法。
