Urinary Glycoprotein Biomarker Discovery for Bladder Cancer Detection Using LC/MS-MS and Label-Free Quantification

Abstract

Background Cancers of the urinary bladder are the fifth most commonly diagnosed malignancy in the US. Early clinical diagnosis of bladder cancer remains a major challenge and the development of non-invasive methods for detection and surveillance is desirable for both patients and health care providers. Approach In order to identify urinary proteins with potential clinical utility we enriched and profiled the glycoprotein component of urine samples using a dual-lectin affinity chromatography and LC-MS/MS platform. Results From a primary sample set obtained from 54 cancer patients and 46 controls a total of 265 distinct glycoproteins were identified with high confidence, and changes in glycoprotein abundance between groups were quantified by a label-free spectral counting method. Validation of candidate biomarker alpha-1-antitrypsin (A1AT) for disease association was performed on an independent set of 70 samples (35 cancer cases) using an ELISA. Increased levels of urinary alpha-1-antitrypsin (A1AT) glycoprotein were indicative of the presence of bladder cancer (p value < 0.0001) and augmented voided urine cytology results. A1AT detection classified bladder cancer patients with a sensitivity of 74% and specificity of 80%. Summary The described strategy can enable higher resolution profiling of the proteome in biological fluids by reducing complexity. Application of glycoprotein enrichment provided novel candidates for further investigation as biomarkers for the non-invasive detection of bladder cancer.

Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD

“…Urine is potentially a rich source for N-and Olinked glycoproteins derived from renal-and distal organs and represents an interesting subproteome for structural charac-terization of human glycoproteins. However, glycoproteomic characterization of urine is lacking and only a few proteomic studies aimed at identifying urinary glycoproteins have been reported (17)(18)(19)(20). In these studies, the glycan moieties were either cleaved off or not studied at all.…”

Targeted High-Throughput Glycoproteomics for Glyco-Biomarker Discovery

“…Top down workflows that incorporate lectin affinity chromatography have been used to identify potential biomarkers in diseases including psoriasis (Plavina et al 2007), hepatocellular carcinoma (Na et al 2009), diabetic nephropathy (Ahn et al 2010) and bladder cancer (Yang et al 2011). Plavina et al depleted the two most abundant plasma proteins, albumin and immunoglobulin, and performed M-LAC consisting of ConA, WGA and JAC to identify numerous tissue leakage proteins present in plasma at low ng/mL concentrations, such as galectin-binding protein 3, which was subsequently verified by ELISA (Plavina et al 2007).…”

“…Ahn et al used M-LAC to capture plasma glycoproteins and found 13 up-regulated and 14 down-regulated glycoproteins in diabetic nephropathy (Ahn et al 2010). Yang et al used ConA and WGA for dual-lectin affinity chromatography to enrich for glycoproteins in urine to identify biomarker candidates for bladder cancer and identified 265 glycoproteins with higher abundance in the cancer group compared to the control group (Yang et al 2011). While there was an overlap of the proteins identified, 240 glycoproteins were uniquely identified by each of the methods.…”

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].…”