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.
authors.affiliation: Department of Chemistry and Biochemistry, 95 Chieftain Way, Florida State University, Tallahassee, Florida 32306, United States.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.forename: Paul A
authors.initials: PA
authors.lastname: Stewart
authors.forename: Qiang
authors.initials: Q
authors.lastname: Cao
authors.forename: Qing-Xiang Amy
authors.initials: QX
authors.lastname: Sang
authors.forename: Leland W K
authors.initials: LW
authors.lastname: Chung
authors.forename: Mark R
authors.initials: MR
authors.lastname: Emmett
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
mesh_terms: D000595:Amino Acid Sequence; D002130:Calcium Phosphates / Q000737:chemistry; D045744:Cell Line, Tumor; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D050684:E2F Transcription Factors / Q000378:metabolism; D005583:Fourier Analysis; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009376:Neoplasms, Hormone-Dependent / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000378:metabolism*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D058570:TOR Serine-Threonine Kinases; D014025:Titanium / Q000737:chemistry
chemical_list: D002130:Calcium Phosphates; D050684:E2F Transcription Factors; D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome; C009495:titanium dioxide; C020243:calcium phosphate; D014025:Titanium; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases