Phosphoproteome of human glioblastoma initiating cells reveals novel signaling regulators encoded by the transcriptome

PLoS One. 2012;7(8):e43398. doi: 10.1371/journal.pone.0043398. Epub 2012 Aug 17.

Abstract

Background: Glioblastoma is one of the most aggressive tumors with poor prognosis. Although various studies have been performed so far, there are not effective treatments for patients with glioblastoma.

Methodology/principal findings: In order to systematically elucidate the aberrant signaling machinery activated in this malignant brain tumor, we investigated phosphoproteome dynamics of glioblastoma initiating cells using high-resolution nanoflow LC-MS/MS system in combination with SILAC technology. Through phosphopeptide enrichment by titanium dioxide beads, a total of 6,073 phosphopeptides from 2,282 phosphorylated proteins were identified based on the two peptide fragmentation methodologies of collision induced dissociation and higher-energy C-trap dissociation. The SILAC-based quantification described 516 up-regulated and 275 down-regulated phosphorylation sites upon epidermal growth factor stimulation, including the comprehensive status of the phosphorylation sites on stem cell markers such as nestin. Very intriguingly, our in-depth phosphoproteome analysis led to identification of novel phosphorylated molecules encoded by the undefined sequence regions of the human transcripts, one of which was regulated upon external stimulation in human glioblastoma initiating cells.

Conclusions/significance: Our result unveils an expanded diversity of the regulatory phosphoproteome defined by the human transcriptome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / metabolism
  • Blotting, Western
  • Brain Neoplasms / genetics
  • Brain Neoplasms / metabolism
  • Brain Neoplasms / pathology
  • Cell Line, Tumor
  • Chromatography, Liquid
  • Epidermal Growth Factor / pharmacology
  • Fibroblast Growth Factors / pharmacology
  • Glioblastoma / genetics
  • Glioblastoma / metabolism
  • Glioblastoma / pathology
  • Humans
  • Isotope Labeling
  • Mass Spectrometry
  • Molecular Sequence Data
  • Neoplastic Stem Cells / drug effects
  • Neoplastic Stem Cells / metabolism*
  • Neoplastic Stem Cells / pathology
  • Phosphopeptides / analysis*
  • Phosphopeptides / genetics
  • Phosphoproteins / analysis*
  • Phosphoproteins / genetics
  • Proteome / analysis*
  • Proteome / genetics
  • Proteomics / methods
  • Sequence Homology, Amino Acid
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Transcriptome / drug effects
  • Transcriptome / genetics

Substances

  • Amino Acids
  • Phosphopeptides
  • Phosphoproteins
  • Proteome
  • Fibroblast Growth Factors
  • Epidermal Growth Factor

Grant support

This work was supported by Cell Innovation Program, Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.