Objective: The aim of this study was to evaluate the differentially secreted protein profile in the urine from patients with clear cell renal cell carcinoma (ccRCC) using mass spectrometry-based methods. Urine composition can reflect kidney physiology and can be used to detect markers for renal diseases. Moreover, characterization of the secretome is likely to assist in the investigation of new drugs for biological targets and diagnose the ccRCC at an early stage.
Methods and materials: Urine samples from patients were divided according to Fuhrman degree (FI-IV), which was associated with the cellular differentiation as good prognosis (GP) and poor prognosis (PP). Healthy individuals were used as the control group (CG). We used both qualitative and quantitative mass spectrometry-based analyses that involved the following approaches: 1-dimensional gel electrophoresis combined with liquid chromatography mass spectrometry in tandem (1DE LC-MS/MS), in-solution digestion combined with label-free 1-dimensional LC-MS(E) (1D LC-MS(E)), and bidimensional gel electrophoresis combined with matrix-assisted laser desorption/ionization time of flight in tandem (2DE MALDI-TOF/TOF) or combined with LC-MS/MS.
Results: All the strategies allowed the identification of 354 proteins from the CG, GP, and PP groups. Qualitative experiments using 1DE LC-MS/MS analysis detected different protein profiles, and 224 proteins were identified in all groups. The label-free MS(E) quantitative analysis identified 113 proteins and generated novel information on secreted protein profiles, including 49 up-secreted proteins in the urine from patients with ccRCC and 40 down-secreted proteins related to the CG. Proteins such as kininogen-1, uromodulin, apolipoprotein D, polyubiquitin, and CD59 glycoprotein were down secreted according to the groups CG>GP>PP. In contrast, apolipoprotein A, fibrinogen, and haptoglobin were up secreted in patient groups. The same expression profile observed for kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin was corroborated by 2DE LC-MS/MS or 2DE MALDI-TOF/TOF analyses. These 2 strategies also showed 13 differentially secreted proteins among the 3 groups.
Conclusions: The proteins kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin presented similar quantitative protein profiles according to MS(E) and 2DE approaches. The latter proteins were up secreted and the former ones were down-regulated. The strategies used proved to be valuable in identifying proteins that were differentially secreted in urine from patients with RCC.
Keywords: Label-free MS(E); Quantitative proteomics; Renal cell carcinoma; Secreted proteins; Urine.
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