Characterization of the phosphoproteome in androgen-repressed human prostate cancer cells by fourier transform ion cyclotron resonance mass spectrometry

Xu Wang, Paul A. Stewart, Qiang Cao, Qing Xiang Amy Sang, Leland W.K. Chung, Mark R. Emmett, Alan G. Marshall

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Androgen-repressed human prostate cancer, ARCaP, grows and is highly metastatic to bone and soft tissues in castrated mice. The molecular mechanisms underlying the aberrant responses to androgen are not fully understood. Here, we apply state-of-the-art mass spectrometry methods to investigate the phosphoproteome profiles in ARCaP cells. Because protein biological phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptides is low, selective enrichment of phosphorylated proteins/peptides is required for mass spectrometric analysis of phosphorylation from complex biological samples. Therefore, we compare the sensitivity, efficiency, and specificity for three established enrichment strategies: calcium phosphate precipitation (CPP), immobilized metal ion affinity chromatography (IMAC), and TiO 2-modified metal oxide chromatography. Calcium phosphate precipitation coupled with the TiO 2 approach offers the best strategy to characterize phosphorylation in ARCaP cells. We analyzed phosphopeptides from ARCaP cells by LC-MS/MS with a hybrid LTQ/FT-ICR mass spectrometer. After database search and stringent filtering, we identified 385 phosphoproteins with an average peptide mass error of 0.32 ± 0.6 ppm. Key identified oncogenic pathways include the mammalian target of rapamycin (mTOR) pathway and the E2F signaling pathway. Androgen-induced proliferation inhibitor (APRIN) was detected in its phosphorylated form, implicating a molecular mechanism underlying the ARCaP phenotype.

Original languageEnglish (US)
Pages (from-to)3920-3928
Number of pages9
JournalJournal of Proteome Research
Volume10
Issue number9
DOIs
StatePublished - Sep 2 2011
Externally publishedYes

Keywords

  • E2F transcription factor
  • FT-MS
  • FTICR
  • LC-MS/MS
  • TiO
  • androgen-induced proliferation inhibitor
  • calcium phosphate precipitation
  • mTOR
  • phosphoproteomics
  • phosphorylation sites
  • prostate cancer

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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