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Detection of Known and Unique Prostate Cancer Biomarkers by SELDI Mass Spectrometry

Detection of Known and Unique Prostate Cancer Biomarkers by SELDI Mass Spectrometry. Bao-Ling Adam, Ph.D. Research Assistant Professor. Department of Microbiology and Molecular Cell Biology, Virginia Prostate Center, Eastern Virginia Medical School, Norfolk, VA 23501. Objective.

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Detection of Known and Unique Prostate Cancer Biomarkers by SELDI Mass Spectrometry

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  1. Detection of Known and Unique Prostate Cancer Biomarkers by SELDI Mass Spectrometry Bao-Ling Adam, Ph.D. Research Assistant Professor Department of Microbiology and Molecular Cell Biology, Virginia Prostate Center, Eastern Virginia Medical School, Norfolk, VA 23501

  2. Objective • Evaluate the ProteinChipTM SELDI-TOF-MS system for: • Simultaneous detection of multiple known prostate cancer-associated biomarkers. • Discovery of unique prostate cancer-associated biomarkers.

  3. Goals • Identify PCA Signature Protein Biomarkers for: • Early Detection of PCA • Diagnosis • Prognosis • Therapeutic Targets

  4. Methods • ProteinChipTM SELDI-TOF-MS: • Chemically Defined Chip Arrays • Lysates of LCM Procured Cells • Serum and Seminal Plasma • SELDI Immunoassay • Single • Multiplex

  5. SELDI Analysis of Protein Bound to Chemical Defined Chip SELDI ProteinChipTM Arrays By surface “chemistries” Normal phase Hydrophobic Anionic, Cationic IMAC NR NR NR Me(II) Me(II) Me(II) SO SO 4 4 SO 4

  6. SELDI ProteinChipTM Analysis of Pure Prostate Cancer Biomarkers Bound to H4 Chip. PSP PAP Intensity PSMA M/Z

  7. Comparison Spectra of Pure PSA from Different Sources. Biodesign a Medix b Intensity Sigma c EVMS d 10000 20000 30000 M/Z

  8. Analysis of LCM Procured Prostate Cell Lysates. Blank Cap PAP PSA NPr Intensity Candidate Biomarker PSMA PCA Stroma 50000 75000 100000 M/Z PSP Intensity M/Z

  9. Identification of PSP, PSA, PAP and PSMA in Seminal Plasma from a PCA Patient. Seminal Plasma PSP PSA PAP PSMA PSA complex M/Z

  10. Identification of PSP, PSA, PAP and PSMA in Serum from a PCA Patient. Serum PSA 2 PSP PSA Complex 4 PSMA M/Z

  11. Identification of Potential Unique Prostate Cancer Biomarkers in Lysates of LCM Procured Prostate Cancer Cells. 6 Candidate Biomarker PCA #1 17970.8 4 Intensity 2 0 NPR #1 NPR #2 NPR #3 PCA #1 PCA #2 PCA #3 35000 20000 30000 25000 M/Z

  12. Identification of Potential Unique Prostate Cancer Biomarkers in Lysates of LCM Procured Prostate Cancer Cells. PCA #1 Candidate Biomarker Intensity PCA #1 PCA #2 PCA #3 NPR #1 NPR #2 NPR #3 3000 4000 5000 6000 7000 M/Z

  13. Identification of a 33 KDa PCA Protein Upregulate in PCA Cells. 28693 # 4 NPr 28602 # 3776 NPr # 3 NPr # 3778 NPr 48526 2 28581 4 0.075 0.15 1.5 48837 0.05 28695 0.1 1 2 0.025 36753 0.05 0.5 48192 0 0 Intensity 0 0 # 4 CaP # 3776 CaP # 5 CaP # 3778 CaP 33492 33311 2 4 0.075 0.15 33563 1.5 0.05 49038 0.1 1 2 0.025 33679 0.05 0.5 48488 0 0 0 0 30000 40000 50000 60000 30000 40000 50000 60000 30000 40000 50000 60000 30000 40000 50000 60000 Mass/Charge

  14. Composite Map of Unique Proteins in Seminal Plasma from Normal and PCA patients. 2 Normal 1 0 Average Intensity 1 PCA 2 Mass/Charge (± 1%)

  15. Identification of Potential Unique Prostate Cancer Biomarkers in Seminal Plasma from Normal and PCA Patients. Candidate Biomarker 0.2 PCA #1 0.15 Intensity 0.1 0.05 0 PCA #1 100000 125000 150000 175000 NPr #1 PCA #2 NPr #2 NPr #3 PCA #3

  16. Identification of Potential Unique Prostate Cancer Biomarkers in Seminal Plasma from Normal and PCA Patients. Candidate Biomarker PCA #2 Intensity PCA #1 NPr #1 PCA #2 NPr #2 NPr #3 PCA #3

  17. Intra and Inter-assay Reproducibility of Spectra Obtained by SELDI ProteinChipTM Analysis of Seminal Plasma. 40 Day 1 67280.3  40Da 28445.4  31Da 30 PAP Albumin Free-PSA 47354.4  43Da 20 10 0 40 Day 2 PAP 67365.8  33Da 28468.0  22Da 30 Albumin 47472.9  62Da Free-PSA 20 Intensity 10 0 40 Day 3 67243.4  37Da 28448.8  18Da 30 PAP Albumin Free-PSA 20 47322.0  48Da 10 0 M/Z

  18. SELDI ProteinChipTM Immunoassay 1. Capture/retention 3. Data analysis Sample + sample Intensity Control M/Z wash + EAM 3 3 3 4 4 4 1 1 1 6 6 6 7 7 7 8 8 8 2 2 2 5 5 5 Put in mass reader Control Ab 2. Detection Bait Ab

  19. Generation of PSA Standard Curve for the SELDI ProteinChipTM Immunoassay. 10 B Free-PSA 8 50 6 30 Intensity 10 4 1 0 2 0 M/Z

  20. PSP Standard Curve for the SELDI ProteinChipTM Immunoassay. C

  21. Detection of PSP and PSA in Seminal Plasma by SELDI ProteinChipTM Immunoassay. PSP  PSP PSA  PSA Intensity PSP PSA  PSP +  PSA  IgG 10000 15000 20000 25000 30000 M/Z

  22. Detection of PSMA and PSA in Seminal Plasma by SELDI ProteinChipTM Immunoassay. PSMA  PSMA PSA  PSA Intensity PSA PSMA  PSMA +  PSA  IgG 10000 60000 80000 40000 M/Z

  23. SELDI ProteinChipTM Analysis of Purified Free-PSA and Complexed PSA (PSA-ACT). a Free PSA Dimer Intensity b (PSA+ACT)+ (PSA+ACT)++ M/Z

  24. Detection of Free-PSA and Complexed PSA (PSA-ACT) by SELDI ProteinChipTM Immunoassay. A  free PSA =  PSA complex Free-PSA Intensity =  Total PSA =  IgG = 30000 75000 85000 20000 M/Z B  free PSA =  PSA complex Intensity = PSA-ACT  Total PSA =  IgG = 30000 75000 85000 20000 M/Z

  25. Detection of Free-PSA and Complexed PSA (PSA-ACT) in Seminal Plasma and Serum by SELDI ProteinChipTM Immunoassay. a Seminal Plasma Free-PSA PSA-ACT = Intensity b Serum Free-PSA PSA-ACT = 25000 30000 35000 80000 90000 M/Z

  26. Conclusions • Four known prostate cancer biomarkers (PSA, PAP, PSP, and PSMA) were successfully identified by SELDI analysis in LCM procured cell lysates, serum, and seminal plasma bound to chemically defined protein chips. • These prostate cancer associated proteins were also successfully identified by single and multiplex SELDI immunoassays. • Both free-PSA and complex (PSA-ACT) forms of PSA were specifically identified by SELDI immunoassays. • The potential of SELDI for the discovery of unique prostate cancer biomarkers was demonstrated.

  27. Collaborators • Eastern Virginia Medical School • Lisa H. Cazares • Lei Gong • Shuhail Nasim, M.D. • Paul F. Schellhammer, M.D. • Antonia Vlahou, Ph.D. • George L. Wright, Jr., Ph.D. • Ciphergen Biosystems, Inc. • Sau-Mei Leung, Ph.D. • Brad Thacher, Ph.D. • Tai-Taung Yip, Ph.D.

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