Development of a strategy for the analysis of metal-binding proteins in urine

The existence of several thousand proteins in urine with concentrations spanning multiple orders of magnitude hinders comparative studies of the urinary proteome. To reduce sample complexity and at the same time enrich for metal binding proteins, with the present study we describe a fractionation strategy for urinary proteins based on the use of immobilized metal chromatography. In brief, urine samples were concentrated by ultrafiltration and protein extracts loaded on Ni super(+2) IMAC columns. Elution of bound proteins was carried out by the use of imidizole. The eluted fractions were subjected to dialysis and subsequently analyzed by 1D and 2D electrophoresis. The resulting protein bands and spots respectively, were identified by LC-MS/MS and subjected to analysis according to Gene Ontology (GO). Multiple metal-binding proteins were identified within the eluted fractions which could not be resolved in the unfractionated starting material. This approach is currently in use for the comparative analysis of metal binding proteins from urine samples from bladder cancer patients and controls.