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Motif-Targeting Phosphoproteome Analysis of Cancer Cells for Profiling Kinase Inhibitors.

Kosuke OgataShunsuke TakagiNaoyuki SugiyamaYasushi Ishihama
Published in: Cancers (2022)
We present a motif-targeting phosphoproteome analysis workflow utilizing in vitro kinase reaction to enrich a subset of peptides with specific primary sequence motifs. Phosphopeptides are enriched and dephosphorylated with alkaline phosphatase, followed by in vitro kinase reaction to phosphorylate substrate peptides with specific primary-sequence motifs. These phosphopeptides are enriched again, TMT-labeled, dephosphorylated to enhance MS-detectability, and analyzed by LC/MS/MS. We applied this approach to inhibitor-treated cancer cells, and successfully profiled the inhibitory spectra of multiple kinase inhibitors. We anticipate this approach will be applicable to target specific subsets of the phosphoproteome using the wide variety of available recombinant protein kinases.
Keyphrases
  • amino acid
  • cancer therapy
  • multiple sclerosis
  • tyrosine kinase
  • protein kinase
  • single cell
  • computed tomography
  • density functional theory
  • binding protein
  • peripheral blood
  • cell free
  • positron emission tomography