Biomarker Detection in Urinary Proteome of Prostate Cancer by Nanoflow LC-MS/MS

Leonora V. Autus-Geniston,1,2 Carlos P. Garcia,2,3 John Donnie A. Ramos,2,3
Alexander O. Tuazon1,4 and Virgilio C. Estanislao5

1Medical and Regulatory Affairs, United Laboratories, Inc., Mandaluyong City, Philippines
2College of Medicine, University of Santo Tomas Graduate School, Espana, Manila
3College of Science, University of Santo Tomas, Espana, Manila
4Philippine General Hospital, University of the Philippines Manila
5University of California-Berkeley, Berkeley, California, USA

Introduction. Urinary proteomics provides a wealth of information in the identification of protein markers associated with various diseases such as in carcinoma. With the increasing incidence of prostate cancer and the lack of sensitivity and specificity of prostate specific antigen, the simultaneous identification of an alternative protein biomarker through urinary proteomics is encouraging. Urine, which has similar proteins with serum, makes it an ideal alternative biofluid wherein the collection is easy and non-invasive.

Methods. Urinary proteins were separated by gradient SDS-PAGE followed by in-gel digestion and organic/buffer peptide extraction. The protein biomarkers in prostate cancer patients and control subjects were identified via LC-MS/MS and submitted to Protein Prospector where the peptide fragmentation of sequence was analyzed and compared with the SwissProt database.

Results. A panel of three protein biomarkers for the early detection of prostate cancer were identified: transthyretin,  hemoglobin subunit alpha and hemoglobin sububit beta. The presence of these three biomarkers is associated with high Gleason scores and TNM stages but not with PSA level. Uromodulin and mannan binding lectin serine protease cancer from BPH. The study also revealed the divergence of the urinary proteome of the cancer patients from the urinary proteome of the control with BPH suggesting the fundamental differences in benign and malignant growth of the prostate epithelial cells. Another highlight of the study was the identification of oxidation of pro63 of transthyretin in patient 3. The proposed role of the post translational modification in pro63 of transthyretinin in the mechanism of prostate carcinogenesis remains to be defined and warrants further study.

Conclusion. Our study was able to establish the homology of urine proteome among the controls and its divergence from the patients afflicted with prostate cancer by simultaneously comparing their urine proteomes leading to the identification of a distinct panel of biomarkers, namely, transthyretin, hemoglobin subunit alpha and hemoglobin subunit beta. Uromodulin and mannan binding lectin serine protease 2 are the additional biomarkers that can distinguish prostate cancer from BPH. Due to limitations in the number of controls and patients, only preliminary findings and their significance were shown. These findings need to be confirmed in future investigations using larger sample size for both the controls and the patients.

Key Words: urinary proteomics, tandem mass spectrometry, nano-high pressure liquid chromatography, prostate cancer, transthyretin