Dataset Information

Feasibility of label-free phosphoproteomics and application to base-line signaling of colorectal cancer cell lines.

ABSTRACT: Data from ProteomeXchange, PXD ID: PXD001550. File: QE2_130117_OPL1013_TiOx_CRC_DLD1_3_130125023714.mzml. Published as part of J Proteomics. 2015 Apr 1. pii: S1874-3919(15)00103-7 . From the Abstract: {{i}} Robust phosphopeptide enrichment methods with minimal fractionation are required to profile signaling network analysis in cancer cell lines and tissues. We assessed performance of single-shot LC-MS/MS label-free phosphoproteomics using TiOx-based phosphopeptide enrichment and report phosphopeptide identification reproducibility (75.8%), depth of identification (6014-6150 phosphopeptides) and reproducibility of label-free quantification (CV 17.8%). Subsequently, we have profiled the baseline global phosphorylation of 8 colorectal cancer (CRC) cell lines representing different CRC prognostic subtypes ... {{/i}}

INSTRUMENT(S): Instrument

ORGANISM(S): Homo_sapiens_viruses, Human

DISEASE(S): Not Available

SUBMITTER: Piersma SR, et al.

PROVIDER: GPM32320014765 | GPMDB |

REPOSITORIES: GPMDB

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Publications

Feasibility of label-free phosphoproteomics and application to base-line signaling of colorectal cancer cell lines.

Piersma Sander R SR Knol Jaco C JC de Reus Inge I Labots Mariette M Sampadi Bharath K BK Pham Thang V TV Ishihama Yasushi Y Verheul Henk M W HM Jimenez Connie R CR

Journal of proteomics 20150401 Pt B

Robust phosphopeptide enrichment methods with minimal fractionation are required to profile signaling network analysis in cancer cell lines and tissues. We assessed performance of single-shot LC-MS/MS label-free phosphoproteomics using TiOx-based phosphopeptide enrichment and report phosphopeptide identification reproducibility (75.8%), depth of identification (6014-6150 phosphopeptides) and reproducibility of label-free quantification (CV 17.8%). Subsequently, we have profiled the baseline glob ...[more]

PMID: 25841592

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Project description:Deleted in Liver Cancer-1 (DLC1), a member of the RhoGAP family of proteins, functions as a tumor suppressor in several cancers including breast cancer. However, its clinical relevance is unclear in breast cancer. In this study, expression of DLC1 was correlated with prognosis using publicly available breast cancer gene expression datasets and quantitative Reverse Transcription PCR in cohorts of Estrogen Receptor-positive (ER+) breast cancer. Mutation and methylation status of DLC1 were assessed in these datasets including The Cancer Genome Atlas (TCGA). To seek further insights in understanding the role of DLC1 in ER+ breast cancer, we developed a knock-in model of DLC1 in T47D breast cancer cells. Label-free global proteomic and TiO2 phosphopeptide enrichment assays were performed and validated using Western blotting. Here, we report that low expression of DLC1 correlates with poor prognosis in patients with ER+ breast cancer with further decrease in metastatic lesions. Analysis of the TCGA data showed that down regulation of DLC1 is not due to methylation or mutations. Stable knock-in of DLC1-full-length inhibits cell growth significantly in vitro compared to its control counterpart. 6726 phosphopeptides were quantified by phosphoproteomics analysis in both conditons, whereas 205 and 122 were unique to DLC1 knock-in and T47D-control cells, respectively. Pathway analysis using DAVID showed the top three significant clusters of differentially identified phosphopeptides involving cell-cell adhesion, mRNA processing and splicing, and transcription regulation. Decreased phosphorylation of epithelial cell transforming sequence 2 (ECT2) at the residue T359, critical for its active conformational change was validated. In conclusion, this data suggests that high expression of DLC1 reduces cell growth and is associated with favorable prognosis. Our results document an inverse relation between ECT2 phosphorylation and DLC1 expression, promising a novel strategy for treating ER+ breast cancer.

Dataset Information

Feasibility of label-free phosphoproteomics and application to base-line signaling of colorectal cancer cell lines.

Publications

Feasibility of label-free phosphoproteomics and application to base-line signaling of colorectal cancer cell lines.

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