Dataset Information

Human CRC cell line baseline phosphoproteomics

ABSTRACT: There is a need for robust phosphopeptide enrichment methods to allow signaling network analysis in cancer cell lines and tissues with minimal fractionation. With recent instrument developments thousands of unique phosphopeptides can be detected by single-shot LC-MS/MS. However, successful phosphoproteomics experiments still rely on efficient phosphopeptide enrichment from a tryptic digest prior to LC-MS/MS analysis. Here we describe a performance assessment of HAMMOC (hydroxyl acid modified metal affinity chromatography) (Sugiyama MCP2007, Kyono, JPR 2008) combined with single shot label-free quantitation at 500 µg peptide input level. We apply the method to profile the baseline phosphorylation landscape of a panel of 8 colorectal cancer (CRC) cell lines. These CRC cell lines represent the 3 CRC subtypes (CCS1, CCS2 and CCS3) as reported by large-scale transcriptome analysis. We report an analysis of the phosphoprotein network and processes enriched in the cell lines representing the poor prognosis CCS3 subtype.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Colon Cancer

SUBMITTER: Sander Piersma

LAB HEAD: Connie Ramona Jimenez

PROVIDER: PXD001550 | Pride | 2015-04-15

REPOSITORIES: Pride

ACCESS DATA

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

Similar Datasets

Project description:Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular functional and structural changes. It requires tight regulation, since disturbances in any of the spermatogenic stages would lead to fertility deficiencies. In order to increase our knowledge of signal transduction during sperm development, we carried out a large-scale identification of the phosphorylation events that occur in the human gonad. Metal oxide affinity chromatography using TiOx combined with LC-MS/MS was conducted to profile the phosphoproteome of human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active and abundant human protein kinases in the testis were predicted both by the phosphorylation status of the kinase activation loop and the number of phosphopeptide spectra identified. The potential function of two of those kinases, cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4(PAK4), has been explored by protein-protein interaction analysis, immunodetection in human and mouse testicular tissue, and functional assay in a human embryonal carcinoma cell line. The co-localization of CDK12 with Golgi markers and probably pro-acrosomal vesicles suggests a potential crucial role of this protein kinase in sperm formation. PAK4 expression has been found limited to human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our data confirm that phosphoregulation by protein kinases is highly active in sperm differentiation, and open a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility, and tumor behavior.

Dataset Information

Human CRC cell line baseline phosphoproteomics

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure