Sample preparation and CE-MS analysis
The standard operating protocols describing urine sample preparation and extraction of peptides followed in this study, have been applied in numerous studies as reviewed previously [29, 30]. The CE-MS analysis was conducted as described in detail by Zurbig et al. [31], utilizing a P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Fullerton, CA, USA) coupled with a Micro-TOF II MS (Bruker Daltonic, Bremen, Germany) instrument. Literary evidence on the advantages of CE-MS analysis in terms of reproducibility, sensitivity, precision and accuracy are extensively available[32]. The relative peptide intensities were normalized based on an internal standard of 29 stable collagen peptides, that can be detected naturally in urine samples of healthy and diseased. This calibration was performed for normalizing the variability in peptide intensities[33]. The resulting peptides and their normalized intensity values were stored in an internal Microsoft SQL database[34], which enabled the comparison of the pre- and post-treatment urinary peptide profiles. For identification of the peptide sequences, MS/MS based analysis by a Dionex Ultimate 3000 RSLS nanoflow system (Dionex, Camberley, UK) or a Beckman P/ACE MDQ CE that was coupled to an Orbitrap Velos MS instrument (Thermo Fisher Scientific Inc., Boston, MA, USA) was performed, as described previously[35].