Systematic verification of bladder cancer-associated tissue protein biomarker candidates in clinical urine specimens
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Cheng-Han Tsai1,*, Yi-Ting Chen1,2,3,4,*, Ying-Hsu Chang5,6,*, Chuen Hsueh2,7, Chung-Yi Liu5, Yu-Sun Chang1,2,8, Chien-Lun Chen9,10 and Jau-Song Yu1,2,11,12
1Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
2Molecular Medicine Research Center, College of Medicine, Chang Gung University, Taoyuan, Taiwan
3Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
4Department of Nephrology, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan, Taiwan
5Division of Urology, Department of Surgery, LinKou Chang Gung Memorial Hospital, Taoyuan, Taiwan
6Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
7Department of Pathology, Chang Gung Memorial Hospital, Linkou, Chang Gung University, College of Medicine, Taoyuan, Taiwan
8Department of Otolaryngology - Head & Neck Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan
9Department of Urology, Chang Gung Memorial Hospital, Taoyuan, Taiwan
10College of Medicine, Chang Gung University, Taoyuan, Taiwan
11Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan
12Research Center for Food and Cosmetic Safety, Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan
*These authors contributed equally to this work
Yi-Ting Chen, email: firstname.lastname@example.org
Jau-Song Yu, email: email@example.com
Chien-Lun Chen, email: firstname.lastname@example.org
Keywords: bladder cancer; biomarker verification; MRM; targeted proteomics; protein quantification
Received: July 02, 2017 Accepted: February 20, 2018 Published: July 20, 2018
Bladder cancer biomarkers currently approved by the Food and Drug Administration are insufficiently reliable for use in non-invasive clinical diagnosis. Verification/validation of numerous biomarker candidates for BC detection is a crucial bottleneck for novel biomarker development. A multiplexed liquid chromatography multiple-reaction-monitoring mass spectrometry assay of 122 proteins, including 118 up-regulated tissue proteins, two known bladder cancer biomarkers and two housekeeping gene products, was successfully established for protein quantification in clinical urine specimens. Quantification of 122 proteins was performed on a large cohort of urine specimens representing a variety of conditions, including 142 hernia, 126 bladder cancer, 67 hematuria, and 59 urinary tract infection samples. ANXA3 (annexin A3) and HSPE1 (heat shock protein family E member 1), which showed the highest detection frequency in bladder cancer samples, were selected for further validation. Western blotting showed that urinary ANXA3 and HSPE1 protein levels were higher in bladder cancer samples than in hernia samples, and enzyme-linked immunosorbent assays confirmed a higher urinary concentration of HSPE1 in bladder cancer than in hernia, hematuria and urinary tract infection. Immunohistochemical analyses showed significantly elevated levels of HSPE1 in tumor cells compared with non-cancerous bladder epithelial cells, suggesting that HSPE1 could be a useful tumor tissue marker for the specific detection of bladder cancer. Collectively, our findings provide valuable information for future validation of potential biomarkers for bladder cancer diagnosis.
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