Identification of precursor forms of free prostate-specific antigen in serum of prostate cancer patients by immunosorption and mass spectrometry.
The structural features of the free prostate-specific antigen (F-PSA) present in human blood have not been clarified up to now, and it is, therefore, not known why F-PSA is not complexed by the protease inhibitors that are present in human blood in large amounts. This lack of information is mainly attributable to the low amount of F-PSA in serum, which makes the isolation and structural characterization very difficult, especially when only limited amounts of individual sera are available. It has now been demonstrated that F-PSA occurs as a mixture of different pro-PSA forms (zymogen forms) in the sera of prostate cancer patients, and that, in some of these sera, a form with the regular NH2 terminus of PSA is present as well. Among the five serum samples investigated, all contained the (-7), (-5), and (-4) pro-PSA forms, whereas the (-1) and (-2) forms were only present in three of them. These three samples also contained the form with the regular NH2 terminus. The (-3) and (-6) pro-PSA forms have not been detected thus far. The F-PSA has been isolated by immunosorption from the individual sera using streptavidin-coated magnetic beads. The pro-PSA forms were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry after producing peptides by endoproteinase from Lysobacter enzymogenes digestion of the SDS-PAGE-separated F-PSA band. The structural identity of the (-7)pro-PSA form was further proven by sequencing of that particular peptide using electrospray ionization quadrupole time-of-flight mass spectrometry.
Peter
J
J
Institut für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany.
Unverzagt
C
C
Krogh
T N
TN
Vorm
O
O
Hoesel
W
W
D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007163:Immunosorbent Techniques; D008297:Male; D010446:Peptide Fragments / Q000032:analysis; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000302:isolation & purification; D011471:Prostatic Neoplasms / Q000097:blood / Q000276:immunology*; D011498:Protein Precursors / Q000097:blood* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010446:Peptide Fragments; D011498:Protein Precursors; D017430:Prostate-Specific Antigen