On-column trypsinization allows for re-use of matrix in modified multiplexed inhibitor beads assay

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 pr...

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Bibliographic Details
Published in:Analytical biochemistry 2017-04, Vol.523, p.10-16
Main Authors: Petrovic, Voin, Olaisen, Camilla, Sharma, Animesh, Nepal, Anala, Bugge, Steffen, Sundby, Eirik, Hoff, Bård Helge, Slupphaug, Geir, Otterlei, Marit
Format: Article
Language:English
Subjects:
Affinity
Biological Assay
Cell signaling
Cell Survival - drug effects
Humans
Inhibitors
Kinase
Mass Spectrometry
Neoplasms - drug therapy
Neoplasms - metabolism
Neoplasms - pathology
Protein Kinase Inhibitors - pharmacology
Protein Kinases - chemistry
Protein Kinases - metabolism
Proteomics - methods
Signal Transduction
Trypsin
Trypsin - pharmacology
Tumor Cells, Cultured
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2017.01.027