Anion exchange chromatographic fractionation of urinary proteins followed by tandem mass spectrometry identifies potential natural inhibitors of calcium oxalate stone

Recent kidney stone research has attempted to define new strategies and therapeutic/preventive targets to reduce the disease prevalence. This study aimed to identify natural inhibitors of calcium oxalate (CaOx) stones in the urine of non-stone/healthy subjects that could serve as novel targets for stone prevention. Urinary proteins were fractionated by diethylaminoethyl anion exchange followed by GigaCap Q-650M column chromatography. Fractionated protein samples (Q1-Q9) were then examined for their CaOx-inhibitory activities. Among these fractions, Q3, Q4, Q5 and Q6 had the most potent inhibitory activities against CaOx crystals. NanoLC-ESI-Qq-TOF MS/MS identified 24-40 proteins from each of these four fractions, in which 16 proteins were detected in all of these potent inhibitory fractions. Interestingly, 20 proteins have been reported with lower urine levels in CaOx stone patients compared with non-stone/healthy subjects. Moreover, 5 proteins (vitronectin, trefoil factor 1, prothrombin, protein AMBP and ceruloplasmin) are the known inhibitors supported by experimental evidence from previous reports. In summary, we have identified a large number of potential natural inhibitors of CaOx stones from normal human urine after fractionation of urinary proteins followed by crystal assays and tandem mass spectrometry. These (possible) novel CaOx stone inhibitors may serve as new targets to prevent CaOx renal stones.