KiC Assay: A quantitative mass spectrometry-based approach

Yadong Huang, Jay J. Thelen

Research output: Chapter in Book/Report/Conference proceedingChapter

12 Scopus citations


Protein phosphorylation is one of the most important posttranslational modifications (PTMs) involved in the transduction of cellular signals. The number of kinases in eukaryotic genomes ranges from several hundred to over one thousand. And with rapidly evolving mass spectrometry (MS)-based approaches, thousands to tens of thousands of phosphorylation sites (phosphosites) have been reported from various eukaryotic organisms, from man to plants. In this relative context, few bona fide kinase-client relationships have been identified to date. To merge the gap between these phosphosites and the cognate kinases that beget these events, comparable large-scale methodologies are required. We describe in detail a MS-based method for identifying kinase-client interactions and quantifying kinase activity. We term this novel Kinase-Client assay, the KiC assay. The KiC assay relies upon the fact that substrate specificities of many kinases are largely determined by primary amino acid sequence or phosphorylation motifs, which consist of key amino acids surrounding the phosphorylation sites. The workflow for detecting kinase-substrate interactions includes four major steps: (1) preparation of purified kinases and synthetic peptide library, (2) in vitro kinase peptide library assay, (3) liquid chromatography (LC)-tandem MS (MS/MS) analysis, and (4) data processing and interpretation. Kinase activity is quantified with the KiC assay by monitoring spectral counts of phosphorylated and unphosphorylated peptides as the readout from LC-tandem mass spectrometry. The KiC assay can be applied as a discovery assay to screen kinases against a synthetic peptide library to find kinase-client relationships or as a targeted assay to characterize kinase kinetics.

Original languageEnglish (US)
Title of host publicationQuantitative Methods in Proteomics
EditorsMarcus Katrin
Number of pages12
StatePublished - 2012

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745


  • In vitro kinase peptide assay
  • Mass spectrometry
  • Multistage activation
  • Phosphoproteomics
  • Protein kinase
  • Protein phosphorylation
  • Spectral counting
  • Workflow


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