TY - JOUR
T1 - Strategy for sensitive and specific detection of molecular forms of PSA based on 2DE and kinetic analysis
T2 - A step towards diagnosis of prostate cancer
AU - Kumar, Vijay
AU - Hassan, Md Imtaiyaz
AU - Singh, Abhay Kumar
AU - Dey, Sharmistha
AU - Singh, Tej P.
AU - Yadav, Savita
N1 - Funding Information:
We appreciate Dr. Sarman Singh, Additional Professor, Department of Laboratory Medicine, AIIMS, New Delhi for arranging to provide human seminal samples. This work supported by financial grants from the Department of Science and Technology (DST), Government of India. We thank the Council of Scientific and Industrial Research (CSIR), New Delhi for the fellowship granted to VK and MIH.
PY - 2009/5
Y1 - 2009/5
N2 - 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.
AB - 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.
KW - Biomarker and tumor proliferation
KW - Peptidic inhibitors
KW - Prostrate specific antigen
KW - Proteomics
KW - Surface plasmon resonance
UR - http://www.scopus.com/inward/record.url?scp=67349156113&partnerID=8YFLogxK
U2 - 10.1016/j.cca.2008.11.020
DO - 10.1016/j.cca.2008.11.020
M3 - Article
C2 - 19118539
AN - SCOPUS:67349156113
SN - 0009-8981
VL - 403
SP - 17
EP - 22
JO - Clinica Chimica Acta
JF - Clinica Chimica Acta
IS - 1-2
ER -