A facile strategy for comprehensive proteome analysis of urine using ultracentrifugation fractionation, solid-phase alkylation based sample preparation and liquid chromatography-mass spectrometry

In this work, a facile strategy with combination of ultracentrifugation fraction, solid-phase alkylation sample preparation and liquid chromatography-mass spectrometry (LC-MS) was developed to achieve comprehensive proteome profiling of urine (CPU). By this strategy, a total of 1659 proteins could be identified using a short LC gradient of ∼1h, 2.3 times more than those (730) obtained from raw urine without fractionation. Compared to the current existing methods for urine sample preparation, not only the analysis time could be greatly shortened by 3–4 times, but also the identification coverage of urine proteome was increased by 130%–160%. Such a method was further combined with label-free quantification for the comparative proteome analysis of urine from IgA nephropathy (IgAN) patients and healthy donors, 227 differentially expressed proteins were identified. In addition to 90 proteins that were reported to be closely related to IgAN, we found that multiple members of solute carrier family 22 (SLC22) were up-regulated in IgAN, which might be related to the disturbance of renal metabolic function in patients. All these results demonstrated that our developed method would provide a promising tool for discovering disease related biomarkers in urine.