Urine samples (5-10 mL) were collected in sterile tubes and held at 4°C for up to 48 h before centrifugation (2,000×g× 20 min at room temperature) and freezing of the supernatant at −70°C. Urine processing, preparation of peptides, extraction, and fractionation are as previously described [18, 19, 24] . Urinary samples were processed by centrifugal filtration at 3,000×g for 20 min at 10°C through Amicon Ultra centrifugal filtration devices (10 kDa cutoff; Millipore, Bedford, MA) preequilibrated with 10 ml Milli-Q water. The filtrate (urine peptidome) containing the low MW naturally occurring peptides was processed with Waters Oasis HLB Extraction Cartridges (Waters Corporation, Milford, MA) and extracted with ethyl acetate. The resulting urine peptide samples were quantified by the 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, as previously described [28] . Three nanomoles peptides were injected on a 100 μm×15 cm C18 reverse-phase column (Michrom) and eluted with a gradient of 5% to 55% acetonitrile over 50 min using a Michrom MS4 HPLC. Twenty-second fractions were collected onto MALDI targets with a Probot fraction collector (LC Packings). A total of 100 fractions were collected and analyzed on 4700 MALDI-TOF/TOF (Applied Biosystems) in MS mode. One microliter of matrix solution containing 4.8 mg/ml α-cyano-4-hydroxycinnamic acid (Agilent Technologies, Palo Alto, CA, USA) and 30 fmol/μl glu-fibrinopeptide (Sigma-Aldrich, St. Louis, MO, USA) was automatically deposited by the Probot on each spot.