Discovery of novel bladder cancer biomarkers by comparative urine proteomics using iTRAQ technology.
A urine sample preparation workflow for the iTRAQ (isobaric tag for relative and absolute quantitation) technique was established. The reproducibility of this platform was evaluated and applied to discover proteins with differential levels between pooled urine samples from nontumor controls and three bladder cancer patient subgroups with different grades/stages (a total of 14 controls and 23 cancer cases in two multiplex iTRAQ runs). Combining the results of two independent clinical sample sets, a total of 638 urine proteins were identified. Among them, 55 proteins consistently showed >2-fold differences in both sample sets. Western blot analyses of individual urine samples confirmed that the levels of apolipoprotein A-I (APOA1), apolipoprotein A-II, heparin cofactor 2 precursor and peroxiredoxin-2 were significantly elevated in bladder cancer urine specimens (n = 25-74). Finally, we quantified APOA1 in a number of urine samples using a commercial ELISA and confirmed again its potential value for diagnosis (n = 126, 94.6% sensitivity and 92.0% specificity at a cutoff value of 11.16 ng/mL) and early detection (n = 71, 83.8% sensitivity and 94.0% specificity). Collectively, our results provide the first iTRAQ-based quantitative profile of bladder cancer urine proteins and represent a valuable resource for the discovery of bladder cancer markers.
Chen
Yi-Ting
YT
Molecular Medicine Research Center, Chang Gung University, Taiwan.
Chen
Chien-Lun
CL
Chen
Hsiao-Wei
HW
Chung
Ting
T
Wu
Chih-Ching
CC
Chen
Chi-De
CD
Hsu
Chia-Wei
CW
Chen
Meng-Chieh
MC
Tsui
Ke-Hung
KH
Chang
Phei-Lang
PL
Chang
Yu-Sun
YS
Yu
Jau-Song
JS
D014408:Biomarkers, Tumor / Q000652:urine*; D016022:Case-Control Studies; D006801:Humans; D009363:Neoplasm Proteins / Q000652:urine; D040901:Proteomics / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis*
D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins