Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled with quantitative proteomics

Abstract

In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity in discriminating bladder cancer from hernia than either marker alone. A marker panel composed by two novel biomarker candidates, SAA4 and proEGF, was first discovered and verified successfully using Western blotting. To the best of our knowledge, the associations of urinary SAA4 and proEGF with bladder tumor and kidney cancer have not been mentioned before. In the present study, we discovered and verified SAA4 and proEGF as potential bladder cancer biomarker for the first time.

Development and Validation of Urine-based Peptide Biomarker Panels for Detecting Bladder Cancer in a Multi-center Study

“…Several of the biomarkers included in the classifiers were previously reported as biomarkers for the detection of urothelial bladder cancer. Apolipoprotein A-I peptide fragments were found up-regulated in both primary and recurrent disease in line with earlier reported findings on this protein in urothelial bladder cancer following application of two-dimensional electrophoresis (2-DE), or iTRAQ/LC-MS/MS-based proteomics analysis (28)(29)(30)(31)(32)(33)(34)(35). In addition, Fibrinogen chains a and b, well known urinary biomarkers for the detection of bladder cancer (30,31,(33)(34)(35), were also found at increased levels in urine from patients with urothelial bladder cancer and included in the classifiers.…”

Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer

“…First morning urine specimens were collected from bladder cancer patients and hernia patients (used as controls for urinary protein biomarker verification studies) on the date of surgery. Clinical specimens were collected as previously described (6,7). Briefly, urine samples were collected in the presence of a protease inhibitor mixture (one tablet/50 ml of urine; Roche, Mannheim, Germany) and sodium azide (1 mM) from hernia and bladder cancer patients.…”

Quantitative body fluid proteomics in medicine — A focus on minimal invasiveness

“…Under normal conditions, urine contains proteins originating from the blood and kidneys [134][135][136][137][138][139], making urine a good source for analysis of diseases affecting the kidney or the urogenital tract; such as kidney failure resulting from high blood pressure and diabetic nephropathy [140,141], prostate cancer [142,143], polycystic kidney disease [144], kidney chronic allograft dysfunction [145], chronic allograft nephropathy [146], congenital obstructive nephropathy [147], lupus nephritis [148], urolithiasis [149], in addition to urinary, renal and bladder cancer [150][151][152][153][154][155][156][157][158][159][160][161]. Besides urogenital and kidney dysfunctions, urinary proteomics has a great potential in biomarker studies of coronary artery atherosclerosis [162,163], obstructive sleep apnea [164], ovarian cancer [165], breast cancer [166] and sepsis [167,168].…”

“…Urine proteome analysis may potentially unravel markers for cancers of urogenital or systemic origin including bladder [150][151][152][153][154]156,157,160,161], prostate [170], renal [158,159], breast [166] and ovarian cancers [165] (Table 7). There has been an increasing interest in developing urine biomarkers for the detection of renal allograft rejection as an alternative to percutaneous needle biopsy, which is costly and associated with significant patient morbidity and mortality [79].…”