Proteomic analysis of urine in patients with intestinal segments transposed into the urinary tract

Abstract

Intestinal segments are used to replace or reconstruct the urinary bladder when it has become dysfunctional or develops life-threatening disease such as cancer. The quality of life in patients with intestinal segments used to either enlarge or completely replace the native bladder is adversely affected by recurrent urinary tract infections, excessive mucus production and the occasional development of malignancy. At present, there is no reliable method of predicting or noninvasively monitoring these patients for the development of these complications. The characterisation of proteins secreted into urine from the transposed intestinal segments could serve as important indicators of these clinical complications. Urine is an ideal source of material in which to search for biomarkers, since it bathes the affected tissues and can be obtained relatively easily by noninvasive methods. The urinary proteome of patients with intestinal segments transposed into the urinary tract is unknown and we present the first global description of the urinary protein profile in these patients. Sample preparation is a critical step in achieving accurate and reliable data. We describe a method to prepare urinary proteins that was compatible with their subsequent analysis using two-dimensional polyacrylamide gel electrophoresis. This method helped to overcome some of the technical problems encountered in analysing urine from this patient cohort. The method was used to analyse urinary proteins recovered from five healthy controls and ten patients with intestinal segments transposed into the urinary tract. Four low molecular weight proteins were found to be present in nine out of ten for the patient group but for none of the healthy controls. The four proteins were identified as lithostathine-1 alpha precursor, pancreatitis associated protein-1 precursor, liver fatty acid binding protein and testis expressed protein-12. The role of these proteins as potential biomarkers of intestinal cell activity within the reconstructed bladder is discussed.

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

“…Two-dimensional electrophoresis (2-DE) of proteins has been the conventional method for biomarker assessment in urological proteomics (33, 34). Irmak et al identified two proteins, orosomucoid (ORM) and zinc-alpha glycoprotein (ZAG) which were increased in urine samples of tumor-bearing patients in comparison with samples from a few healthy volunteers (17), and Pinero et al used 2D-DIGE coupled with mass spectrometry to identify regenerating protein-1 (Reg-1) and keratin 10 as being associated with bladder cancer (35).…”

Bladder Cancer Associated Glycoprotein Signatures Revealed by Urinary Proteomic Profiling

“…Two-dimensional electrophoresis (2-DE) of proteins has been the conventional method for biomarker assessment in urological proteomics, 14,15 and investigators have performed a systematic evaluation of sample preparation methods for gelbased human urinary proteomes. 16 A near-standard 2-D map 17 and high-resolution 2-D gels 18 of urological tissues have contributed to the construction of a 2-D database.…”

Discovery of Urinary Biomarkers

“…Nephrologists and urologists have begun to use mass spectrometry-based methods for discovery of potential disease biomarkers for a number of renal diseases including diabetic glomerulopathy (14,16,37,69), acute renal injury (22), IgA nephropathy (15), Fanconi syndrome of various causes (29,31,70), idiopathic glomerulopathy (13), congenital obstructive nephropathy (39,71), vesicoureteral reflux (72), interstitial cystitis (73), and urinary bladder reconstruction (74).…”