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].