建立多重反應監測質譜法以用於非小型細胞肺癌中的突變EGFR定量

Mutations in the kinase domain of epidermal growth factor receptor (EGFR) trigger ligand-independent proliferation pathways in non-small-cell lung cancer (NSCLC). Tyrosine kinase inhibitors (TKIs) that specifically block the mutation-induced signalings are prescribed for patients with genetic EGFR mutations and achieve an overall objective response rate of 71% in Asia. However, the responses vary from complete (2.3%), partial response (68.9%) to stable disease (20.5%), which suggests a poor correlation between genetic EGFR mutations and TKI responses. Because TKIs target directly on EGFR proteins, we hypothesized that protein-level detection of EGFR mutations might provide better prediction of TKI response. We aimed to develop a multiple reaction monitoring-mass spectrometry (MRM-MS) assay for multiplexed and absolute quantitation of EGFR mutations, including single amino acid substitutions (G719A and L858R) and exon 19 deletion (del 746A-750D), at protein level. We used NSCLC cell lines as model for assay development. EGFR proteins were purified by immunoprecipitation, digested with Lys-C and Glu-C, and analyzed by LC-MRM-MS. A list of 14 peptides and 112 transitions were selected and optimized for MRM-MS analysis. We achieved a maximum of 16.4 -fold and median of 0.28 fold improvements for the transition intensities after optimization of collision energy. We measured 20fmol of del E746-A750 from 4mg of PC9 cell lysate, 6.4fmol of del E746-A750 from 2mg of CL68 cell lysate, 12.3fmol of L858R from 2mg of H3255 cell lysate and 56.4fmol of G719A from 4mg of CL97 cell lysate. In the future, we will apply the MRM-MS analysis in NSCLC patient samples and we expect that our assay will improve the prediction power for TKI response.