Comparison of different depletion strategies for improving resolution of the human urine proteome

Background: Urine, being an ultrafiltrate of plasma, is a rich source for biomarker discovery. Since potential new disease markers are often present in low concentrations, a prefractionation/enrichment step could be useful in the discovery process. To enhance the detection of low-abundance proteins, three immuno-affinity depletion approaches were evaluated. Methods: To remove the most abundant proteins from a human urine sample, GenWay(TM) Spin IgY-12 kit, HPLC Agilent Hu-PL7 and a home-made column vs. human serum albumin [immuno-affinity column (IAC)] were compared. Quantification of total proteins, 2-D gel electrophoresis (2-DE), Progenesis gel images analysis and mass spectrometric proteins identification were applied to evaluate these strategies. Results: Reproducibility of depletion columns, by estimating protein content of unbound fractions, were: 343+/-20.0 mu g, 5.8%; 186.3+/-13.3 mu g, 7.2%; 292+/-20.6 mu g, 8.8% [mean+/-standard deviation (SD), CV%], for GenWay(TM), Agilent and IAC methods, respectively. To isolate urinary protein after depletion, ethanol precipitation provided the highest recovery (80%). Applying 2-DE and Progenesis analysis, the number of spots visualized on the gels was 468+/-21, 331+/-7, 368+/-22 and 304+/-7 (mean+/-SD) for GenWay(TM), Agilent, IAC, and the undepleted urine pool sample, respectively, with a significant difference p < 0.001 compared to the GenWay procedure. Conclusions: The sequential procedure of urine samples using multi-protein immuno-affinity depletion represents a valid tool for simplifying 2-DE analysis of the urine proteome. Particularly, the GenWay(TM) kit followed by ethanol precipitation was found to be the most efficient method for exploring the urine proteome. Clin Chem Lab Med 2010;48:531-5.