title: Quantitative analysis of prostate specific antigen isoforms using immunoprecipitation and stable isotope labeling mass spectrometry.
abstract: Prostate specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa), but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia (BPH). Recently, proPSAs, comprised of native proPSA, as well as truncated proPSA forms, [-2] proPSA, [-5] proPSA, and [-7] proPSA, have been shown to be better diagnostic targets than PSA for PCa. Stable isotope labeling-multiple reaction monitoring mass spectrometry (SIL/MRM-MS) has been frequently used to measure low-abundance biomarkers in tissues and biofluids, owing to its high sensitivity and specificity, simplicity, and multiplexing capability. In this study, we have developed and optimized a strategy using immunoprecipitation in conjunction with SIL/MRM-MS assay which is capable of sensitive and accurate quantification of proPSA in serum. Since serum and plasma are by far the most complex biological fluids, the immunoprecipitation workflow was optimized to achieve sufficient sensitivity, efficiencies of protein purification with immunoaffinity depletion were determined. The developed strategy can detect proPSA and PSA with a limit of detection (LOD) and limit of quantitation (LOQ) at nanogram per milliliter levels, corresponding to a concentration 6 orders-of-magnitude lower than the most abundant serum proteins. Furthermore, the simultaneous measurement of multiple biomarkers, including the mature and precursor forms of PSA, can be achieved in a single multiplexed analysis using LC/MRM-MS. The strategy demonstrated here provides an attractive alternative to ELISAs or RIAs for the reliably measurement of proPSA to improve the specificity of PCa diagnosis.
authors.affiliation: Department of Chemistry, National Taiwan Normal University , Taipei 11677 Taiwan.
authors.forename: Yi-Ting
authors.initials: YT
authors.lastname: Chen
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Tuan
authors.forename: Hsiao-Wei
authors.initials: HW
authors.lastname: Chen
authors.forename: I-An
authors.initials: IA
authors.lastname: Wei
authors.forename: Min-Yuan
authors.initials: MY
authors.lastname: Chou
authors.forename: Han-Min
authors.initials: HM
authors.lastname: Chen
authors.forename: Yu-Chang
authors.initials: YC
authors.lastname: Tyan
authors.forename: Sung-Fang
authors.initials: SF
authors.lastname: Chen
mesh_terms: D001681:Biological Assay; D001798:Blood Proteins / Q000032:analysis*; D002853:Chromatography, Liquid; D005260:Female; D064368:Healthy Volunteers; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D007553:Isotope Labeling; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis*; D017430:Prostate-Specific Antigen / Q000097:blood*; D011471:Prostatic Neoplasms / Q000097:blood / Q000175:diagnosis*; D020033:Protein Isoforms; D011863:Radioimmunoassay
chemical_list: D001798:Blood Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D017430:Prostate-Specific Antigen