Identification of Urinary CD44 and Prosaposin as Specific Biomarkers of Urinary Tract Infections in Children With Neurogenic Bladders

Forster, Catherine S; Haffey, Wendy D; Bennett, Michael; Greis, Kenneth D; Devarajan, Prasad

Five patients with unequivocal UTIs and 5 patients with UTC with positive urine cultures and no clinical symptoms were included in the initial identification of candidate markers. Sample volumes were reduced with a 3-kDa filter into 1× Laemmli buffer. The protein concentrations were measured using the Pierce 660 nm Protein Assay kit (Thermo Scientific) as this assay is both compatible with higher concentrations of detergents and maintains a greater linear range than standard Coomassie dye-binding assays (eg, Bradford assay). The samples were then run in a 1D, 1.5 cm 4% to 12% Bis-Tris gel using MOPS (3-(morpholino)propanesulfonic acid) running buffer. The regions of each lane between the well and the dye front were excised for trypsin digestion. The resulting peptides were extracted and the control lanes pooled together. The recovery of the digested peptides in each sample was determined via Nanodrop analysis. The resulting peptides from each sample were tagged with the indicated iTRAQ reagent using the vendor instructions. Samples for each iTRAQ set were mixed 1:1:1:1 based on the peptide Nanodrop reading and loaded onto a Sciex 5600+ nanoflow LC-mass spectrometry system, as Forster et al 3 previously described. 15 Each 4-plex set contained one common control sample made from an equal protein mixture of all samples in the cohort. Measuring protein ratios against a single common control then allows for cross comparison of the relative protein levels among all the samples in the cohort. ProteinPilot software (Sciex) was used to identify the proteins and determine the relative quantitation from each run. A merged search of all runs identified the full scope of proteins detected across all groups. The ProteinPilot data were then processed through Protein Alignment Template software from Sciex. Proteins with a significant fold-change between the UTI and UTC groups (those at >50% change in the average abundance across groups) were identified as proteins of interest. Operators were blinded to sample group until the analysis stage, when unblinding was necessary to interpret the results.