Urine and Catheter Specimen Extraction and Protein Solubilization for Proteomics

The catheter materials were latex in the case of eight patients and silicone (patient P7). The color and turbidity of urine specimens and salt crystallization inside and on the surface of catheter pieces were noted. To obtain a UP from a sample, an aliquot was thawed, adjusted to 20°C, and, if acidic, neutralized with 1 M Tris–HCl (pH 8.1) to a pH of ~6.5 to 7.5 and centrifuged at 3,200 g for 15 min. UPs were aliquoted for rDNA and protein extractions and spare samples in ratios of ~10, 45, and 45%, respectively. Urethral catheter pieces were extracted in two steps. Submerged in 100 mM sodium acetate (pH 5.5), 20 mM sodium metaperiodate, and 300 mM NaCl, catheter pieces were agitated in an ultrasonic water bath for 10 min at 20°C. The supernatants were recovered and concentrated followed by two solubilization cycles of the pellets with a denaturing SED solution [1% sodium dodecyl sulfate (SDS) (vol/vol), 5 mM EDTA and 50 mM DTT] including 3-min heat treatment at 95°C. Two supernatants were recovered that we termed CB₁ (Na-acetate buffer) and CB₂ (SED solution). CB₁ and CB₂ fractions were separately analyzed via LC-MS/MS. Raw data were merged for database searches as described in the following section. UP samples were solubilized in SED solution as described previously (13, 27). Centrifugation steps were performed using Ultrafree-4 filter units (10 kDa MWCO) to collect large peptides and proteins in the concentrates. Peptides with low M_r values such as neutrophil defensin 1 (4 kDa) were also retained. Solubilized UP and CB samples were subjected to filter-aided sample preparation (FASP) (55) adapted to 100 μg protein for digestion with sequencing-grade trypsin (50:1 ratio) in Vivacon 10k filters (Sartorius AG, Germany) (13). FASP-processed peptide mixtures were desalted using the Stage-Tip method (56) and lyophilized for LC-MS/MS analysis.