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Lotan Y., Svatek R. S., Nandi A., Gurnani P.
Presented on March, 22 2007
INTRODUCTION & OBJECTIVES: PSA has a positive predictive value of only 25-30% secondary to its low specificity. Recent advances in mass spectrometry instrumentation, bioinformatics, and sample preparation techniques have enabled the development of the field of clinical proteomics. The aim of the study is to identify reliable serum proteomic biomarkers to discriminate between the presence or absence of prostate cancer in men.
MATERIAL & METHODS: Between 2/2004 and 9/2005, blood was obtained from 253 patients with elevated PSA prior to prostate biopsy or prostate surgery (for benign or malignant conditions) and men with known prostate cancer prior to radical prostatectomy. Serum samples were processed by using ProXPRESSIONTM Biomarker Enrichment Kits and directly spotted on single-use MALDIchipTM Target plates (PerkinElmer). Mass spectra were acquired using a prOTOFTM 2000 matrix-assisted laser desorption/ionization orthogonal time-of-flight (MALDI O-TOF) MS interfaced with TOFWorksTM software (PerkinElmer/SCIEX). Samples were run in triplicate. Progenesis PG600 (Nonlinear Dynamics Ltd., UK.) was used to analyze the spectrum peaks. For identification, a protein of interest (m/z 5901.7) was enriched using the enrichment kits (ProXPRESSION). ProXPRESSIONTM kits and IMAC30 columns were used to enrich for a ~12 kDa peak and then gel purified using SDS-PAGE and Sypro Ruby. We sequenced the protein associated with this peak after in gel tryptic digestion and MS/MS-based sequencing.
RESULTS: Our study group included 179 patients with prostate cancer with a mean age of 61.1 and mean PSA of 9.1 ng/ml. There were 74 controls with a mean age of 63.9 years and mean PSA levels of 6.08 ng/ml. We identified 12 monoisotopic peaks that had significant differences between cancer and non-cancer specimens with p values less than 0.05 in all 3 replicates of the serum samples. The analysis also identified 4 peaks (3190, 3206, 3261 and 3277) that had a significant difference in marker intensities between prostate cancer patients with features of aggressive cancer (serum PSA ≥20 ng/ml, Gleason sum ≥8, metastases to lymph nodes, and/or extraprostatic extension) versus those without (p<0.05). One of the discriminatory peaks identified was a peak of 5901.7 Da. The peak was a multiply charged (+2H) species from a parent molecule of 11,803.4 Da in molecular weight. We identified this protein to be serum retinol binding protein (PRO2222, 17.9 kDa MW, dominant tryptic fragment LIVHNGYCDGR, GI number 7770173), which has an expected mass at 17.9 kDa. This peak was found to be expressed (i.e. by MS peak intensities) at 36% less intensity in cancer than in normals (p=0.06).
CONCLUSIONS: Proteomics offers the potential for identification of new serum markers in detection and prognostication of prostate cancer. Our study identifies several discriminatory peaks that differentiate patients with prostate cancer from benign disease. We demonstrate the feasibility of marker identification by sequencing retinol binding protein which has a decreased expression in patients with prostate cancer.
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