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 Emmett, Alan G. Marshall

Research output: Contribution to journalArticle

7 Citations (Scopus)

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

Fingerprint

Cyclotrons
Cyclotron resonance
Phosphorylation
Fourier Analysis
Androgens
Mass spectrometry
Phosphopeptides
Mass Spectrometry
Prostatic Neoplasms
Fourier transforms
Cells
Ions
Metals
Affinity chromatography
Ion chromatography
Peptides
Phosphoproteins
Mass spectrometers
Sirolimus
Chromatography

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Cite this

Characterization of the phosphoproteome in androgen-repressed human prostate cancer cells by fourier transform ion cyclotron resonance mass spectrometry. / Wang, Xu; Stewart, Paul A.; Cao, Qiang; Sang, Qing Xiang Amy; Chung, Leland W K; Emmett, Mark; Marshall, Alan G.

In: Journal of Proteome Research, Vol. 10, No. 9, 02.09.2011, p. 3920-3928.

Research output: Contribution to journalArticle

Wang, Xu ; Stewart, Paul A. ; Cao, Qiang ; Sang, Qing Xiang Amy ; Chung, Leland W K ; Emmett, Mark ; Marshall, Alan G. / Characterization of the phosphoproteome in androgen-repressed human prostate cancer cells by fourier transform ion cyclotron resonance mass spectrometry. In: Journal of Proteome Research. 2011 ; Vol. 10, No. 9. pp. 3920-3928.
@article{e912c10379af4c868e6adee0869437a7,
title = "Characterization of the phosphoproteome in androgen-repressed human prostate cancer cells by fourier transform ion cyclotron resonance mass spectrometry",
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.",
keywords = "androgen-induced proliferation inhibitor, calcium phosphate precipitation, E2F transcription factor, FT-MS, FTICR, LC-MS/MS, mTOR, phosphoproteomics, phosphorylation sites, prostate cancer, TiO",
author = "Xu Wang and Stewart, {Paul A.} and Qiang Cao and Sang, {Qing Xiang Amy} and Chung, {Leland W K} and Mark Emmett and Marshall, {Alan G.}",
year = "2011",
month = "9",
day = "2",
doi = "10.1021/pr2000144",
language = "English (US)",
volume = "10",
pages = "3920--3928",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "9",

}

TY - JOUR

T1 - Characterization of the phosphoproteome in androgen-repressed human prostate cancer cells by fourier transform ion cyclotron resonance mass spectrometry

AU - Wang, Xu

AU - Stewart, Paul A.

AU - Cao, Qiang

AU - Sang, Qing Xiang Amy

AU - Chung, Leland W K

AU - Emmett, Mark

AU - Marshall, Alan G.

PY - 2011/9/2

Y1 - 2011/9/2

N2 - 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.

AB - 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.

KW - androgen-induced proliferation inhibitor

KW - calcium phosphate precipitation

KW - E2F transcription factor

KW - FT-MS

KW - FTICR

KW - LC-MS/MS

KW - mTOR

KW - phosphoproteomics

KW - phosphorylation sites

KW - prostate cancer

KW - TiO

UR - http://www.scopus.com/inward/record.url?scp=80052433685&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=80052433685&partnerID=8YFLogxK

U2 - 10.1021/pr2000144

DO - 10.1021/pr2000144

M3 - Article

VL - 10

SP - 3920

EP - 3928

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 9

ER -