https://scholars.lib.ntu.edu.tw/handle/123456789/558540
標題: | Integrating site-specific peptide reporters and targeted mass spectrometry enables rapid substrate-specific kinase assay at the nanogram cell level | 作者: | Reyes A.J.F. Kitata R.B. dela Rosa M.A.C. Wang Y.-T. Lin P.-Y. PAN-CHYR YANG Friedler A. Yitzchaik S. Chen Y.-J. |
關鍵字: | Multiple reaction monitoring mass spectrometry; Phosphorylation; Substrate-specific kinase activity | 公開日期: | 2021 | 出版社: | Elsevier B.V. | 卷: | 1155 | 起(迄)頁: | 338341 | 來源出版物: | Analytica Chimica Acta | 摘要: | 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 |
URI: | https://www.scopus.com/inward/record.uri?eid=2-s2.0-85101739282&doi=10.1016%2fj.aca.2021.338341&partnerID=40&md5=5c4561680ed5b8f588bf9d00b9d811d6 https://scholars.lib.ntu.edu.tw/handle/123456789/558540 |
ISSN: | 0003-2670 | DOI: | 10.1016/j.aca.2021.338341 | SDG/關鍵字: | 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 |
顯示於: | 醫學系 |
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