Urine 2DE proteome analysis in healthy condition and kidney disease

Abstract

Urine is a source of potential markers of disease. In the context of renal disease, urine is particularly important as it may directly reflect kidney injury. Current markers of renal dysfunction lack both optimal specificity and sensitivity, and improved technologies and approaches are needed. There is no clear consensus about the best sample pretreatment procedure for 2DE analysis of the urine proteome. Sample pretreatment conditions spots resolution and detection sensitivity, critically. As a first goal, we exhaustively compared eight different sample cleaning and protein purification methodologies for 2DE analysis of urine from healthy individuals. Oasis® HLB cartridges allowed the detection of the highest number of low molecular weight proteins; while PD10 desalting columns resulted in the highest number of detected spots in the high molecular weight area. Sample pretreatment strategies were also explored in the context of proteinuria, a clinical condition often associated to renal damage. Testing of urine samples from 13 patients with hypertension or kidney disease and different levels of proteinuria identified Oasis® HLB cartridge purification in combination with albumin depletion by ProteoPrep kit as the best option for urine proteome profiling from patients with proteinuric (> 30 mg/L albumin in urine) renal disease.

Contribution of proteomics to the management of vascular disorders

“…However, technical/ methodological issues have hindered urinary proteomics from contributing significantly to our pathophysiological understanding of CVDs. Although proteinuria is an established risk factor for cardiovascular morbidity and mortality [26], the analysis of lessabundant and naturally existing urinary proteins and peptides in proteinuric patients still remains a challenge [27,28]. Serum and plasma have been the focus of extensive proteomics studies for decades [29].…”

Contributions of immunoaffinity chromatography to deep proteome profiling of human biofluids

“…Furthermore, by utilizing immunodepletion coupled to HPLC-Chip-MS/MS detection, He et al identified 1641 urinary proteins with high confidence and provided a panel comprising 18 urinary proteins to assess the human health status from urine samples of 100 male and 100 female healthy donors [144]. Martin-Lorenzo et al evaluated two immunodepletion approaches including ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit (Sigma) and MARS Hu-14, and they demonstrated that using OasisR HLB cartridge purification in combination with albumin depletion by ProteoPrep kit as the best option for urine proteome profiling from patients with proteinuric renal disease [145]. Magagnotti et al compared three depletion strategies including IgY, MARS and a home-made depletion column for improving coverage of the human urine proteome, and showed IgY depletion followed by ethanol precipitation was the most efficient method for exploring urine proteome [146].…”

Quantitative mass spectrometry of urinary biomarkers

“…The 2DE method is a primary technique that has been widely used in urinary proteomics [29–31]. In this gel-based method, urinary proteins are resolved in the first-dimension based on their isoelectric point (pI) followed by resolution based on molecular weight in the second-dimension.…”

“…The important step before the gel separation is urine concentration. Multiple protocols have been developed to concentrate and purify urinary proteins including lyophilization, precipitation, ultracentrifugation, and centrifugal filtration [11, 18, 29, 32–35]. Analysis of 2DE images is performed using computer-based platforms.…”