No abstract

In typical proteomic analysis, trypsin digestion is one of the most time-consuming steps. Conventional proteomic sample preparation methods use an overnight trypsin digestion method. In this study, we compared high-pressure cycling technology (PCT) during enzyme digestion for proteome analysis to the conventional method. We examined the effect of PCT on enzyme activity at temperatures of 25, 37, and 50 o C. Although a fast digestion (1 h) was used for the standard protein mixture analysis, the PCT-assisted method with urea showed better results for protein sequence coverage and the number of peptides identified compared with the conventional method. There was no significant difference between temperatures for PCT-assisted digestion; however, we selected PCT-assisted digestion with urea at 25 o C as an optimized method for fast enzyme digestion, based on peptide carbamylation at these conditions. The optimized method was used for stem cell proteome analysis. We identified 233, 264 and 137 proteins using the conventional method with urea at 37 o C for 16h, the PCT-assisted digestion with urea at 25 o C for 1 h, and the non-PCT-assisted digestion with urea at 25 o C for 1 h, respectively. A comparison of these results suggests that PCT enhanced the enzyme digestion by permitting better access to cleavage sites on the proteins.

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.