| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 106 === Protein phosphorylation plays an important role to regulate signal transduction pathways. Phosphorylation alteration may occur due to changes at protein expression level or site-specific phosphorylation stoichiometry, defined as the percentage of phosphorylation on specific site of a protein. Only direct measurement of the phosphorylation stoichiometry can unambiguously reveal whether the signal-induced alteration is regulated by protein abundance or by upstream kinase/phosphatase activity.
Using EGFR signaling pathway in non-small cell lung cancer as a model study, we develop a quantitative proteomic method to measure the phosphorylation stoichiometry on EGFR and its downstream substrates in response to EGF and TKI treatment. The method integrates SILAC labeling, motif-targeting kinase reaction, immobilized metal affinity chromatography (IMAC) and LC-MS/MS analysis. The cells were cultured with heavy SILAC isotopes (13C615N4Arg/13C615N2Lys). After protein digestion, the kinase reaction was performed by recombinant EGFR under the 18O-ATP; thus, EGFR substrates can be recognized by 18O-labeling. Finally, the IMAC is utilized for phosphopeptide enrichment, followed by LC-MS/MS analysis. The phosphopeptides with heavy SILAC labeling and 18O-labeling represent the initially unphosphorylated form of potential EGFR substrate sites, while its corresponding 16O-labeled phosphopeptides represent initially phosphorylated form. The phosphorylation stoichiometry can be calculated by the ratio of 16O-phosphopeptide/(16O-phosphopeptide+ 18O-phosphopeptide).
Total of 13,104 phosphopeptides from 4590 proteins were identified. Among 4425 quantified proteins, we obtained stoichiometry of 5,426 phosphopeptides by detecting their 18O-labeled peaks. The result revealed 95 reported EGFR-related proteins and stoichiometry of 49 proteins were successfully measured. In the NSCLC pathway, many EGFR downstream proteins and its sites such as Src pS225, pY419, Shc1 pY427, RASA1 pY460, STAT1 pY170, MAPK pY204 were identified. Upon EGF activation and TKI treatment, we measured altered stoichiometry of many known or predicted EGFR substrates, such as Shc1(Y427), Erk1(Y204), Erk2(Y187), and Src(Y419). The results were verified by western-blotting experiment. Besides EGFR kinase, we also applied CK2 and MAPK kinases to analyze their substrates in the NSCLC pathways. The results also obtained stoichiometry of CK2 and MAPK substrates in NSCLC downstream pathway such as STAT1/3 pS727, AKT1A1 pS211, pS212 and RAF1 S301, which phosphorylation sequence also matched to the kinase motif.
In summary, our new method revealed the first map of phosphorylation stoichiometry of a signaling pathway at the basal level and under stimuli, which provides new insight on the phosphorylation-mediated signaling pathway for better understanding the site-specific TKI response in NSCLC. The method can be generally applied to other sample types such as cells or tissue, body fluids.
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