Strategy for sensitive and specific detection of molecular forms of PSA based on 2DE and kinetic analysis: a step towards diagnosis of prostate cancer.
BACKGROUND
Prostate specific antigen (PSA) present in human seminal fluid exists in many isoforms due to different sequence, glycosylation pattern and polypeptide length. Its presence in various stages of cancer has already been reported.


METHODS
We identified 8 forms of PSA followed by purification of 3 forms. We compared their binding affinities to designed synthetic peptides, by using surface plasmon resonance (SPR). PSA forms were purified using various chromatographic procedures and characterized by 2D gel electrophoresis (2DE), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).


RESULTS
The heterogeneity of purified PSA was demonstrated by 2DE analysis. The peptides were designed on the basis of cleavage map of insulin-like growth factor binding protein 3 (IGFBP-3) subsequently, these peptides showed the binding affinities in the range of 10(-5) to 10(-12) M. Out of 7 peptides, VLLH showed maximum affinity to intact PSA, while FLSYK showed maximum affinity to cleaved forms of PSA. On the other hand, FLSYK in the presence of zinc showed higher affinity to intact PSA. These peptides showed higher affinity to PSA compared to zinc, which is a known inhibitor of PSA.


CONCLUSIONS
This study reports purification and characterization of 3 forms of PSA simultaneously. Furthermore, we have reported specific peptides for different forms of PSA which are either responsible for prostate cancer (PCa) or benign prostatic hyperplasia (BPH). Our study aids the existing information on categorizing the molecular heterogeneity of PSA.
Kumar
Vijay
V
Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India.
Hassan
Md Imtaiyaz
MI
Singh
Abhay Kumar
AK
Dey
Sharmistha
S
Singh
Tej P
TP
Yadav
Savita
S
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D006965:Hyperplasia / Q000175:diagnosis; D007527:Isoenzymes / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017430:Prostate-Specific Antigen / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000175:diagnosis* / Q000201:enzymology*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
D007527:Isoenzymes; D010455:Peptides; D017430:Prostate-Specific Antigen