title: Analytical platform evaluation for quantification of ERG in prostate cancer using protein and mRNA detection methods.
abstract: BACKGROUND
  The established methods for detecting prostate cancer (CaP) are based on tests using PSA (blood), PCA3 (urine), and AMACR (tissue) as biomarkers in patient samples. The demonstration of ERG oncoprotein overexpression due to gene fusion in CaP has thus provided ERG as an additional biomarker. Based on this, we hypothesized that ERG protein quantification methods can be of use in the diagnosis of prostate cancer.
  
  
  METHODS
  An antibody-free assay for ERG3 protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERG negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment). Enzyme-linked immunosorbent assay (ELISA), western blot, NanoString, and qRT-PCR were also used in the analysis of these samples.
  
  
  RESULTS
  Recombinant ERG3 protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine. The sensitivity of the in-house ELISA was similar to the PRISM-SRM assay, with detection of 30 pg of purified recombinant ERG3 protein and 10,000 VCaP cells. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 100 VCaP cells, in comparison to NanoString methodologies which detected ERG from 10,000 cells.
  
  
  CONCLUSIONS
  Based on this data, we propose that the detection of both ERG transcriptional products with RNA-based assays, as well as protein products of ERG using PRISM-SRM assays, may be of clinical value in developing diagnostic and prognostic assays for prostate cancer given their sensitivity, specificity, and reproducibility.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. jintang.he@pnnl.gov.
authors.forename: Jintang
authors.initials: J
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authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. athena.schepmoes@pnnl.gov.
authors.forename: Athena A
authors.initials: AA
authors.lastname: Schepmoes
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. tujin.shi@pnnl.gov.
authors.forename: Tujin
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authors.lastname: Shi
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. chaochao.wu@pnnl.gov.
authors.forename: Chaochao
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authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.fillmore@pnnl.gov.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Fillmore
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. yuqian.gao@pnnl.gov.
authors.forename: Yuqian
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. dick.smith@pnnl.gov.
authors.forename: Richard D
authors.initials: RD
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authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. weijun.qian@pnnl.gov.
authors.forename: Wei-Jun
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authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. karin.rodland@pnnl.gov.
authors.forename: Karin D
authors.initials: KD
authors.lastname: Rodland
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. tao.liu@pnnl.gov.
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authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. dave.camp@pnnl.gov.
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.affiliation: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. arastogi@cpdr.org.
authors.forename: Anshu
authors.initials: A
authors.lastname: Rastogi
authors.affiliation: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. stan@cpdr.org.
authors.forename: Shyh-Han
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authors.affiliation: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. wyan@cpdr.org.
authors.forename: Wusheng
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authors.lastname: Yan
authors.affiliation: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. amohamed@cpdr.org.
authors.forename: Ahmed A
authors.initials: AA
authors.lastname: Mohamed
authors.affiliation: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. whuang@cpdr.org.
authors.forename: Wei
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authors.affiliation: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. sbanerjee@cpdr.org.
authors.forename: Sreedatta
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authors.lastname: Banerjee
authors.affiliation: Cancer Biomarkers Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD, USA. kaganj@mail.nih.gov.
authors.forename: Jacob
authors.initials: J
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authors.affiliation: Cancer Biomarkers Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD, USA. ss1a@nih.gov.
authors.forename: Sudhir
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authors.lastname: Srivastava
authors.affiliation: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. dgmcleod@verizon.net.|Urology Service, Walter Reed National Military Medical Center, Bethesda, MD, USA. dgmcleod@verizon.net.
authors.forename: David G
authors.initials: DG
authors.lastname: McLeod
authors.affiliation: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. ssrivastava@cpdr.org.
authors.forename: Shiv
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authors.lastname: Srivastava
authors.affiliation: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. gpetrovics@cpdr.org.
authors.forename: Gyorgy
authors.initials: G
authors.lastname: Petrovics
authors.affiliation: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. adobi@cpdr.org.
authors.forename: Albert
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authors.lastname: Dobi
authors.affiliation: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. asrinivasan@cpdr.org.
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mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000652:urine; D012333:RNA, Messenger; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism / Q000652:urine; D000071230:Transcriptional Regulator ERG
chemical_list: C072312:ERG protein, human; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG