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Phosphoproteome of ecto-ATP synthase blockade in tumor xenograft tissues
ATP synthase is crucial for ATP synthesis in living cells. Recently, ATP synthase was found not only in mitochondria but also on the extracellular surface, named as ectopic ATP synthase (ecto-ATP synthase). ATP synthase inhibitor is a potential drug candidate to fight cancer by blocking the ecto-ATP synthase of cells. In this study, we applied dynamic phosphoproteomics to elucidate the molecular responses to ecto-ATP synthase blockade.
Sample Processing Protocol
The tissue samples were obtained from a CL1-0 xenograft model of our previous study, which successfully affirmed the inhibitory effect of citreoviridin on tumor growth in vivo. The immunodeficient mice carrying subcutaneous CL1-0 tumors were abdominally injected with citreoviridin or DMSO and sacrificed when the control tumors reached a size of 1000 mM3. Proteins from the dissected tissues were digested, dimethyl-labeled, and processed to phosphopeptide enrichment. Phosphopeptides were analyzed by LC-MS/MS with biological replicates.
Data Processing Protocol
Peptide identification was performed by Mascot v2.3 (Matrix Science, London, UK) against both human and mouse database of Swiss-Prot human database version 83. The search criteria used in this study were trypsin specificity, fixed modification of carbamidomethyl (C), variable modifications of oxidation (M) and phosphorylation (STY), and allowing up to 2 missed cleavages. The precursor mass tolerance was 10 ppm and the fragment ion tolerance was 0.1 Da. Data from each nanoLC-MS/MS run was searched individually. Peptides were considered identified if the Mascot score was over 99% confidence limit based on the significance threshold (p < 0.01) and at least three successive y- or b-ions with an additional two and more y-, b- and/or precursor-origin neutral loss ions were observed, based on the error-tolerant peptide sequence tag concept. These criteria gave a false discovery rate of approximately 1% at peptide level as evaluated using the target-decoy strategy. Peptide was quantified by Mass Navigator version 1.3 (Mitsui Knowledge Industry, Tokyo, Japan) according to the intensity of centroids of each labeled peptide.
Hu CW, Hsu CL, Wang YC, Ishihama Y, Ku WC, Huang HC, Juan HF. Temporal phosphoproteome dynamics induced by an ATP synthase inhibitor citreoviridin. Mol Cell Proteomics. 2015 Oct 26. pii: mcp.M115.051383 PubMed: 26503892
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