title: An efficient method for native protein purification in the selected range from prostate cancer tissue digests.
abstract: BACKGROUND
  Prostate cancer (CP) cells differ from their normal counterpart in gene expression. Genes encoding secreted or extracellular proteins with increased expression in CP may serve as potential biomarkers. For their detection and quantification, assays based on monoclonal antibodies are best suited for development in the clinical setting. One approach to obtain antibodies is to use recombinant proteins as immunogen. However, the synthesis of recombinant protein for each identified candidate is time-consuming and expensive. It is also not practical to generate high quality antibodies to all identified candidates individually. Furthermore, non-native forms (e.g., recombinant) of proteins may not always lead to useful antibodies. Our approach was to purify a subset of proteins from CP tissue specimens for use as immunogen.
  
  
  METHODS
  In the present investigation, ten cancer specimens obtained from cases scored Gleason 3+3, 3+4 and 4+3 were digested by collagenase to single cells in serum-free tissue culture media. Cells were pelleted after collagenase digestion, and the cell-free supernatant from each specimen were pooled and used for isolation of proteins in the 10-30 kDa molecular weight range using a combination of sonication, dialysis and Amicon ultrafiltration. Western blotting and mass spectrometry (MS) proteomics were performed to identify the proteins in the selected size fraction.
  
  
  RESULTS
  The presence of cancer-specific anterior gradient 2 (AGR2) and absence of prostate-specific antigen (PSA)/KLK3 were confirmed by Western blotting. Proteomics also detected AGR2 among many other proteins, some outside the selected molecular weight range, as well.
  
  
  CONCLUSIONS
  Using this approach, the potentially harmful (to the mouse host) exogenously added collagenase was removed as well as other abundant prostatic proteins like ACPP/PAP and AZGP1 to preclude the generation of antibodies against these species. The paper presents an optimized scheme for convenient and rapid isolation of native proteins in any desired size range with minor modifications.
authors.affiliation: Department of Urology, University of Washington, Seattle, WA 98195, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA. rumana_ahmad@yahoo.co.in.
authors.forename: Rumana
authors.initials: R
authors.lastname: Ahmad
authors.affiliation: Biological Science Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Carrie D
authors.initials: CD
authors.lastname: Nicora
authors.affiliation: Biological Science Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Shukla
authors.affiliation: Biological Science Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.affiliation: Biological Science Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
authors.affiliation: Department of Urology, University of Washington, Seattle, WA 98195, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Alvin Y
authors.initials: AY
authors.lastname: Liu
mesh_terms: D014408:Biomarkers, Tumor / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D006801:Humans; D007610:Kallikreins / Q000302:isolation & purification; D008297:Male; D009088:Mucoproteins; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D015513:Oncogene Proteins; D017430:Prostate-Specific Antigen / Q000302:isolation & purification; D011471:Prostatic Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry
chemical_list: C502296:AGR2 protein, human; D014408:Biomarkers, Tumor; D009088:Mucoproteins; D009363:Neoplasm Proteins; D015513:Oncogene Proteins; D011506:Proteins; C549870:KLK3 protein, human; D007610:Kallikreins; D017430:Prostate-Specific Antigen
keywords: Prostate cancer proteins (CP proteins); anterior gradient 2 (AGR2); cancer biomarkers; proteomic analysis; purification of 10–30 kDa proteins