| Literature DB >> 28167071 |
Voin Petrovic1, Camilla Olaisen1, Animesh Sharma2, Anala Nepal1, Steffen Bugge3, Eirik Sundby4, Bård Helge Hoff3, Geir Slupphaug2, Marit Otterlei5.
Abstract
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.Entities:
Keywords: Affinity; Cell signaling; Inhibitors; Kinase; MS; Trypsin
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Year: 2017 PMID： 28167071 DOI： 10.1016/j.ab.2017.01.027
Source DB: PubMed Journal: Anal Biochem ISSN： 0003-2697 Impact factor: 3.365