title: Identification of prostate-specific antigen (PSA) isoforms in complex biological samples utilizing complementary platforms.
abstract: Measurements of the prostate-specific antigen (PSA) levels in blood are widely used as diagnostic, predictive and prognostic marker of prostate disease. The selective detection of molecular forms of PSA can contribute clinically to meaningful enhancements of the conventional PSA-test. As it is plausible that an in-depth search for structural variants of PSA gene products may increase our ability to discriminate distinct patho-biological basis and stages of prostate diseases, we have developed a multi-step protocol comprising gel-based methods followed by mass spectrometric identification. Our current aim was to provide a comprehensive identification of PSA variants occurring in seminal fluid. We provide a proof-of-principle for this multiple step analytical approach to identify multiple PSA variants from complex biological samples that revealed distinct molecular characteristics. In addition, sequence-annotated protein bands in SDS-PAGE gels were compared to those detected by Western blots, and by monitoring the enzymatic activity in zymogram gels, using gelatin as a substrate. The high accuracy annotations were obtained by fast turnaround MALDI-Orbitrap analysis from excised and digested gel bands. Multiple PSA forms were identified utilizing a combination of MASCOT and SEQUEST search engines.
authors.affiliation: Div. Clinical Protein Science & Imaging, Dept. of Measurement Technology and Industrial Electrical Engineering, Lund University, BMC C13, SE-221 84 Lund, Sweden. akos.vegvari@elmat.lth.se
authors.forename: Akos
authors.initials: A
authors.lastname: Végvári
authors.forename: Melinda
authors.initials: M
authors.lastname: Rezeli
authors.forename: Charlotte
authors.initials: C
authors.lastname: Welinder
authors.forename: Johan
authors.initials: J
authors.lastname: Malm
authors.forename: Hans
authors.initials: H
authors.lastname: Lilja
authors.forename: György
authors.initials: G
authors.lastname: Marko-Varga
authors.forename: Thomas
authors.initials: T
authors.lastname: Laurell
mesh_terms: D000595:Amino Acid Sequence; D003720:Densitometry / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011467:Prostate / Q000378:metabolism*; D017430:Prostate-Specific Antigen / Q000096:biosynthesis*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012661:Semen / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010455:Peptides; D017430:Prostate-Specific Antigen