Integrating site-specific peptide reporters and targeted mass spectrometry enables rapid substrate-specific kinase assay at the nanogram cell level
Journal
Analytica Chimica Acta
Journal Volume
1155
Pages
338341
Date Issued
2021
Author(s)
Reyes A.J.F.
Kitata R.B.
dela Rosa M.A.C.
Wang Y.-T.
Lin P.-Y.
Friedler A.
Yitzchaik S.
Chen Y.-J.
Abstract
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. ? 2021 The Authors
Subjects
Multiple reaction monitoring mass spectrometry; Phosphorylation; Substrate-specific kinase activity
SDGs
Other Subjects
Cell culture; Cells; Data acquisition; Digital storage; Drug products; Enzymes; Mass spectrometry; Peptides; Phosphorylation; Absolute quantification; Multiple reaction monitoring; Multiplexed analysis; Non small cell lung cancer; Phosphoproteomics; Proof of concept; Reproducibilities; Substrate peptides; Substrates; HDGF S165 peptide; NRD1 S94 peptide; peptide derivative; phosphotransferase; protein tyrosine kinase inhibitor; RALY S135 peptide; unclassified drug; heterogeneous nuclear ribonucleoprotein group C; peptide; RALY protein, human; accuracy; Article; biological monitoring; cancer cell line; cancer resistance; cell level; cell lysate; controlled study; enzyme activity; enzyme analysis; enzyme assay; enzyme phosphorylation; enzyme specificity; feasibility study; human; human cell; mass spectrometry; non small cell lung cancer; PC-9 cell line; phenotype; phosphoproteomics; reproducibility; lung tumor; mass spectrometry; non small cell lung cancer; phosphorylation; Carcinoma, Non-Small-Cell Lung; Heterogeneous-Nuclear Ribonucleoprotein Group C; Humans; Lung Neoplasms; Mass Spectrometry; Peptides; Phosphorylation; Reproducibility of Results
Publisher
Elsevier B.V.
Type
journal article