Project PXD000701

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Summary

Title

Phosphoproteome of ecto-ATP synthase blockade in tumor xenograft tissues

Description

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.

Contact

Chia-Wei Hu, Institute of molecular and cellular biology
Hsueh-Fen Juan, Systems Biology Lab, Department of Life Science, National Taiwan University, Taiwan ( lab head )

Submission Date

21/01/2014

Publication Date

06/11/2015

Publication

    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

Assay

Showing 1 - 4 of 4 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 34307 Xenograft_tissue 1_run1 379 1632 443 30038 1394
2 34306 Xenograft_tissue 1_run2 403 1642 454 28393 1409
3 34305 Xenograft_tissue 2_run1 323 1061 372 28196 821
4 34304 Xenograft_tissue 2_run2 301 1057 349 26674 842