Development of a urine protein preparation method for 2‐D gel analysis and selection of osteoporotic biomarkers

Abstract

Biomarker development for osteoporosis from human urine was carried out by optimizing urine protein preparation methods and 2‐D gel analysis method through proteomic protein identification tools including mass spectrometry and bioinformatics. Preparation of urine proteins for reproducible high resolution 2‐D gel analysis has been a troublesome topic in urine proteomic analysis. We developed a reproducible pretreatment method for urine protein preparations by ultrafiltration, dialysis, and desalting. This method efficiently removes interfering molecules for IEF analysis and improves resolution and reproducibility in 2‐D gel analysis. Using this improved protein preparation method we could obtain good quality 2‐D gels for the comparison of protein profiles between normal and patient urine samples. Urine samples from human osteoporosis patients and osteoporotic animal model from ovariectomy were used to develop biomarkers for osteoporosis. We could selected several osteoporosis associated biomarkers, among which vitamine D binding protein, vitronectin, acid‐glycoprotein‐alpha were confirmed by Western blot analysis. The results suggest that those biomarker proteins can be utilized for the development of diagnostic kits detecting osteoporosis simply from urine. The study was supported by a grant from Korean Ministry of Health, Welfare and Family Affairs.