Integrating site-specific peptide reporters and targeted mass spectrometry enables rapid substrate-specific kinase assay at the nanogram cell level

Aaron James F. Reyes, Reta Birhanu Kitata, Mira Anne C. dela Rosa, Yi Ting Wang, Pei Yi Lin, Pan Chyr Yang, Assaf Friedler, Shlomo Yitzchaik, Yu Ju Chen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Dysregulation of phosphorylation-mediated signaling drives the initiation and progression of many diseases. A substrate-specific kinase assay capable of quantifying the altered site-specific phosphorylation of its phenotype-dependent substrates provides better specificity to monitor a disease state. We report a sensitive and rapid substrate-specific kinase assay by integrating site-specific peptide reporter and multiple reaction monitoring (MRM)-MS platform for relative and absolute quantification of substrate-specific kinase activity at the sensitivity of nanomolar kinase and nanogram cell lysate. Using non-small cell lung cancer as a proof-of-concept, three substrate peptides selected from constitutive phosphorylation in tumors (HDGF-S165, RALY-S135, and NRD1-S94) were designed to demonstrate the feasibility. The assay showed good accuracy (<15% nominal deviation) and reproducibility (<15% CV). In PC9 cells, the measured activity for HDGF-S165 was 3.2 ± 0.2 fmol μg−1 min−1, while RALY-S135 and NRD1-S94 showed 4- and 20-fold higher activity at the sensitivity of 25 ng and 5 ng lysate, respectively, suggesting different endogenous kinases for each substrate peptide. Without the conventional shotgun phosphoproteomics workflow, the overall pipeline from cell lysate to MS data acquisition only takes 3 h. The multiplexed analysis revealed differences in the phenotype-dependent substrate phosphorylation profiles across six NSCLC cell lines and suggested a potential association of HDGF-S165 and NRD1-S94 with TKI resistance. With the ease of design, sensitivity, accuracy, and reproducibility, this approach may offer rapid and sensitive assays for targeted quantification of the multiplexed substrate-specific kinase activity of small amounts of sample.

Original languageAmerican English
Article number338341
JournalAnalytica Chimica Acta
StatePublished - 22 Apr 2021

Bibliographical note

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© 2021 The Authors


  • Multiple reaction monitoring mass spectrometry
  • Phosphorylation
  • Substrate-specific kinase activity


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