Petrovic Voin, Olaisen Camilla, Sharma Animesh, Nepal Anala, Bugge Steffen, Sundby Eirik, Hoff Bård Helge, Slupphaug Geir, Otterlei Marit
Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
The Proteomics and Metabolomics Core Facility (PROMEC) at NTNU, Faculty of Medicine, Trondheim, Norway.
Anal Biochem. 2017 Apr 15;523:10-16. doi: 10.1016/j.ab.2017.01.027. Epub 2017 Feb 3.
The Multiplexed Inhibitor Bead (MIB) assay is a previously published quantitative proteomic MS-based approach to study cellular kinomes. A rather extensive procedure, need for multiple custom-made kinase inhibitors and an inability to re-use the MIB-columns, has limited its applicability. Here we present a modified MIB assay in which elution of bound proteins is facilitated by on-column trypsinization. We tested the modified MIB assay by analyzing extract from three human cancer cell lines treated with the cytotoxic drugs cisplatin or docetaxel. Using only three immobilized kinase inhibitors, we were able to detect about 6000 proteins, including ∼40% of the kinome, as well as other signaling, metabolic and structural proteins. The method is reproducible and the MIB-columns are re-usable without loss of performance. This makes the MIB assay a simple, affordable, and rapid assay for monitoring changes in cellular signaling.
多重抑制剂珠（MIB）分析是一种先前已发表的基于质谱的定量蛋白质组学方法，用于研究细胞激酶组。该方法流程较为繁琐，需要多种定制的激酶抑制剂，且MIB柱无法重复使用，这些因素限制了其应用。在此，我们提出一种改良的MIB分析方法，通过柱上胰蛋白酶消化促进结合蛋白的洗脱。我们通过分析用细胞毒性药物顺铂或多西他赛处理的三种人类癌细胞系的提取物，对改良的MIB分析方法进行了测试。仅使用三种固定化激酶抑制剂，我们就能检测到约6000种蛋白质，包括约40%的激酶组，以及其他信号转导、代谢和结构蛋白。该方法具有可重复性，且MIB柱可重复使用而不损失性能。这使得MIB分析成为一种用于监测细胞信号转导变化的简单、经济且快速的分析方法。
