Multiplexed quantification of 63 proteins in human urine by multiple reaction monitoring-based mass spectrometry for discovery of potential bladder cancer biomarkers

• Published in: Journal of proteomics Vol. 75; no. 12; pp. 3529 - 3545
• Main Authors: Chen, Yi-Ting, Chen, Hsiao-Wei, Domanski, Dominik, Smith, Derek S., Liang, Kung-Hao, Wu, Chih-Ching, Chen, Chien-Lun, Chung, Ting, Chen, Min-Chi, Chang, Yu-Sun, Parker, Carol E., Borchers, Christoph H., Yu, Jau-Song
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 27.06.2012
• Subjects: adiponectin; Amino Acid Sequence; apolipoprotein A-II; Biomarker verification; biomarkers; Biomarkers, Tumor; Biomarkers, Tumor - chemistry; Biomarkers, Tumor - urine; Bladder cancer; bladder diseases; chemistry; complement; diagnosis; Feasibility Studies; ferroxidase; genetic variation; genome; hematuria; hernia; Humans; mass spectrometry; Mass Spectrometry - methods; methods; Molecular Sequence Data; Multiple reaction monitoring; Multiplexed quantitation; Neoplasm Proteins; Neoplasm Proteins - chemistry; Neoplasm Proteins - urine; patients; Peptide Mapping; Peptide Mapping - methods; prothrombin; Reproducibility of Results; Sensitivity and Specificity; urinary bladder neoplasms; Urinary Bladder Neoplasms - diagnosis; Urinary Bladder Neoplasms - urine; Urinary proteome; Urinary tract infection/hematuria; urine; LOD; Urinary tract infection/hematuria; Multiple reaction monitoring; NT; APOA2; APOA1; MRM; UTI; HU; LC–MS/MS; LgEs; Bladder cancer; Multiplexed quantitation; HgEs; LOQ; CV; HgAs; XIC; Biomarker verification; Urinary proteome; SIS
• ISSN: 1874-3919; 1876-7737
• DOI: 10.1016/j.jprot.2011.12.031

Three common urological diseases are bladder cancer, urinary tract infection, and hematuria. Seventeen bladder cancer biomarkers were previously discovered using iTRAQ — these findings were verified by MRM-MS in this current study. Urine samples from 156 patients with hernia (n=57, control), bladder cancer (n=76), or urinary tract infection/hematuria (n=23) were collected and subjected to multiplexed LC–MRM/MS to determine the concentrations of 63 proteins that are normally considered to be plasma proteins, but which include proteins found in our earlier iTRAQ study. Sixty-five stable isotope-labeled standard proteotypic peptides were used as internal standards for 63 targeted proteins. Twelve proteins showed higher concentrations in the bladder cancer group than in the hernia and the urinary tract infection/hematuria groups, and thus represent potential urinary biomarkers for detection of bladder cancer. Prothrombin had the highest AUC (0.796), with 71.1% sensitivity and 75.0% specificity for differentiating bladder cancer (n=76) from non-cancerous (n=80) patients. The multiplexed MRM-MS data was used to generate a six-peptide marker panel. This six-peptide panel (afamin, adiponectin, complement C4 gamma chain, apolipoprotein A-II precursor, ceruloplasmin, and prothrombin) can discriminate bladder cancer subjects from non-cancerous subjects with an AUC of 0.814, with a 76.3% positive predictive value, and a 77.5% negative predictive value. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry. [Display omitted] ► Urinary concentrations of 63 proteins in 156 samples were determined with LC–MRM/MS. ► Patients with bladder cancer (BC), hernia, or UTI/hematuria were studied. ► Twelve proteins showed higher levels in the BC group than in the other two groups. ► Seventeen BC biomarkers discovered by iTRAQ were verified using LC–MRM/MS. ► A six-peptide biomarker panel for BC was found that gave an AUC of 0.814.