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 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.
Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
Voin
V
Petrovic
Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
Camilla
C
Olaisen
The Proteomics and Metabolomics Core Facility (PROMEC) at NTNU, Faculty of Medicine, Trondheim, Norway.
Animesh
A
Sharma
Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
Anala
A
Nepal
Department of Chemistry, Faculty of Natural Sciences and Technology, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
Steffen
S
Bugge
Department of Chemistry and Material Technology, Faculty of Natural Sciences and Technology, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
Eirik
E
Sundby
Department of Chemistry, Faculty of Natural Sciences and Technology, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
Bård Helge
BH
Hoff
The Proteomics and Metabolomics Core Facility (PROMEC) at NTNU, Faculty of Medicine, Trondheim, Norway.
Geir
G
Slupphaug
Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway. Electronic address: marit.otterlei@ntnu.no.
Marit
M
Otterlei
D001681:Biological Assay; D002470:Cell Survival / Q000187:drug effects*; D006801:Humans; D013058:Mass Spectrometry; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D014357:Trypsin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D014357:Trypsin
Affinity; Cell signaling; Inhibitors; Kinase; MS; Trypsin