Abstract
Urinary proteins have been implicated as inhibitors of kidney stone formation (urolithiasis). As a proximal fluid, prefiltered by the kidneys, urine is an attractive biofluid for proteomic analysis in urologic conditions. However, it is necessary to correct for variations in urinary concentration. In our study, individual urine samples were normalized for this variation by using a total protein to creatinine ratio. Pooled urine samples were compared in two independent experiments. Differences between the urinary proteome of stone formers and nonstone-forming controls were characterized and quantified using label-free nano-ultraperformance liquid chromatography high/low collision energy switching analysis. There were 1063 proteins identified, of which 367 were unique to the stone former groups, 408 proteins were unique to the control pools, and 288 proteins were identified for comparative quantification. Proteins found to be unique in stone-formers were involved in carbohydrate metabolism pathways and associated with disease states. Thirty-four proteins demonstrated a consistent >twofold change between stone formers and controls. For ceruloplasmin, one of the proteins was shown to be more than twofold up-regulated in the stone-former pools, this observation was validated in individuals by enzyme-linked immunosorbent assay. Moreover, in vitro crystallization assays demonstrated ceruloplasmin had a dose-dependent increase on calcium oxalate crystal formation. Taken together, these results may suggest a functional role for ceruloplasmin in urolithiasis.
Highlights
Kidney stone disease is a major clinical and economic burden for health care systems around the world
Using ELISA, we investigated whether ceruloplasmin levels in individuals would corroborate the proteomic data generated from pooled samples
In some patients kidney stones can result in kidney failure
Summary
Sample Collection and Patients—This study analyzed urine samples from 57 patients with radiologically confirmed urinary tract stones and 57 patients with unrelated benign urological conditions. Mass Spectrometry and Data Analysis—Tryptic digests of fractionated urinary proteins were subjected to nano-ultraperformance liquid chromatography-tandem mass spectrometry analysis (nano-UPLCMS/MS) using a 75 m I.D. 25 cm C18 column, 1.7 m particle size and a 90 min gradient: 2% to 45% solvent B (solvent A: 99.9% H2O, 0.1% FA; solvent B: 99.9% acetonitrile, 0.1% FA) on a Waters NanoAcquityTM UPLC system coupled to a Waters QTOFpremierTM tandem mass spectrometer (Waters), as described previously [15]. For crystallization experiments 100 l of the stock solution A was transferred into each well of the 96-well plate To this 100 l of the stock solution B was added resulting in final assay concentrations of 4 mM calcium chloride, 0.5 mM sodium oxalate, 200 mM sodium chloride, and 10 mM sodium acetate, pH 6.
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