Despite attractiveness of urine for biomarker discovery for systemic and renal diseases, the confounding effect of the high abundance plasma proteins in urine, and a lack of optimization of urine protein recovery methods are bottlenecks for urine proteomics. Three methods were performed and compared for percentage protein yield, yield consistency, ease and cost of analysis: (i) organic solvent precipitation, (ii) dialysis/lyophilization, and (iii) centrifugal filtration. Urine samples were subjected to an immunoaffinity column to deplete high abundance proteins. Difference gel electrophoresis was performed to assess use of depletion strategy for detection of low abundance proteins. Urine from healthy volunteers (n = 10) and kidney transplant recipients with proteinuria (n = 11) were used. Centrifugal filtration performed best for analysis ease and yield consistency. Highest percentage yield was obtained from dialysis/lyophilization but was laborious and residual salt interfered with subsequent gel electrophoresis. Organic solvent precipitation was inexpensive, but suffered from varying yield consistency. Increased spot intensity for some low abundance and previously undetected proteins were noted after depletion of high abundance proteins. In conclusion, we compare the pros and cons of different protein recovery methods and reveal an increase in the dynamic range of protein detection after depletional strategy that could be critical for biomarker discovery, particularly with reference to processing human study samples from clinical trials.

Objective Necrotising enterocolitis (NEC) is a major source of neonatal morbidity and mortality. The management of infants with NEC is currently complicated by our inability to accurately identify those at risk for progression of disease prior to the development of irreversible intestinal necrosis. We hypothesised that integrated analysis of clinical parameters in combination with urine peptide biomarkers would lead to improved prognostic accuracy in the NEC population. Design Infants under suspicion of having NEC (n=550) were prospectively enrolled from a consortium consisting of eight university-based paediatric teaching hospitals. Twenty-seven clinical parameters were used to construct a multivariate predictor of NEC progression. Liquid chromatography/mass spectrometry was used to profile the urine peptidomes from a subset of this population (n=65) to discover novel biomarkers of NEC progression. An ensemble model for the prediction of disease progression was then created using clinical and biomarker data. Results The use of clinical parameters alone resulted in a receiver-operator characteristic curve with an area under the curve of 0.817 and left 40.1% of all patients in an ‘indeterminate’ risk group. Three validated urine peptide biomarkers (fibrinogen peptides: FGA1826, FGA1883 and FGA2659) produced a receiver-operator characteristic area under the curve of 0.856. The integration of clinical parameters with urine biomarkers in an ensemble model resulted in the correct prediction of NEC outcomes in all cases tested. Conclusions Ensemble modelling combining clinical parameters with biomarker analysis dramatically improves our ability to identify the population at risk for developing progressive NEC.

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Introduction Human urine is a complex matrix of proteins, endogenous peptides, lipids, and metabolites. The level of any or all of these components can reflect the pathophysiological status of an individual especially of the kidney at the time of urine collection. The naturally occurring endogenous urinary peptides which are thought to be the product of several proteolytic and degradation processes may provide clinically useful biomarkers for different renal and systemic diseases. Materials and MethodsTo examine if specific differences in the urinary peptidome (<10 kDa) occur at the time of acute renal transplant rejection (AR), we undertook a study of urine samples collected from biopsy-proven AR (n=10), stable graft function (n=10), and healthy normal control (n=10). The peptides (<10 kDa) were extracted and fractionated with high-performance liquid chromatography followed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric (MS) analysis. Results We identified 54 endogenous peptides, including multiple peptides for Tamm-Horsfall protein (UMOD). A panel of peptides are identified which discriminate renal transplant patients with AR from stable graft. We have shown that liquid chromatography followed by MALDI is a useful tool to identify potential biomarkers, which after verification with larger patient cohort can be used as a noninvasive monitoring tool for renal transplant rejection.