Improvement of resolution in 2-D gel analysis by simple pre-treatment of human urine with EDTA


Regardless of thermodynamic stability, less complex proteome than plasma, convenient and noninvasive sampling of urine, reproducibility and comparability of the data among laboratories are challenges for the urine proteome analysis. Pretreatment of urine with 10 mM EDTA in conjunction with desalting methods to remove interfering molecules significantly improved the presentation of urine proteome on 2-D gels. Statistical analysis of the average ratio of the spot numbers on 2-D gels between EDTA-treated and non-treated samples were 1.35 ± 0.167, 1.26 ± 0.091, and 1.24 ± 0.095 for dialysis, ultrafiltration, and desalting column, respectively, with the p-value of 5.85 × 10–6 for the overall comparison. This result came from the fact that EDTA-treated samples showed better resolutions with less streaks and clearer spots on 2-D gels than the control samples. The results suggest that a simple and non-fractionating EDTA-treatment can be employed to the procedures for the high resolution 2-D gel analysis and other urinary proteomic analysis.

Share and Cite:

Lee, J. , Lee, J. and Lim, J. (2012) Improvement of resolution in 2-D gel analysis by simple pre-treatment of human urine with EDTA. Open Journal of Clinical Diagnostics, 2, 40-43. doi: 10.4236/ojcd.2012.22008.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Pisitkun, T., Johnstone, R. and Knepper, M.A. (2006) Discovery of urinary biomarkers. Molecular and Cellular Proteomics, 5, 1760-1771. doi:10.1074/mcp.R600004-MCP200
[2] Thongboonkerd, V. (2008) Urinary proteomics: towards biomarker discovery, diagnostics and prognostics. Molecular BioSystems, 4, 810-815. doi:10.1039/b802534g
[3] Raimondo, F., Morosi, L., Chinello, C., Magni, F. and Pitto, M. (2011) Advances in membranous vesicle and exosome proteomics improving biological understanding and biomarker discovery. Proteomics, 11, 709-720. doi:10.1002/pmic.201000422
[4] Lee, R.L., Monigatti, F., Briscoe, A.C., Waldon, Z., Freeman, M.R. and Steen, H. (2008) Optimizing sample handling for urinary proteomics. Journal of Proteome Research, 7, 4022-4030. doi:10.1021/pr800301h
[5] Molina, L., Salvetat, N., Ameur, R.B., Peres, S., Sommerer, N., Jarraya, F., Ayadi, H., Molina, F. and Granier, C. (2011) Analysis of the variability of human normal urine by 2D-GE reveals a “public” and a “private” proteome. Journal of Proteomics, 75, 70-80. doi:10.1016/j.jprot.2011.06.031
[6] Putnam, D.F. (1971) Composition and concentrative properties of human urine. NASA contractor report.
[7] Thongboonkerd, V. (2007) Practical points in urinary proteomics. Journal of Proteome Research, 6, 3881-3890. doi:10.1021/pr070328s
[8] Thongboonkerd, V., Chutipongtanate, S. and Kanlaya, R. (2006) Systematic evaluation of sample preparation methods for gel-based human urinary proteomics: Quantity, quality, and variability. Journal of Proteome Research, 5, 183-191. doi:10.1021/pr0502525
[9] Theodorescu, D. and Mischak, H. (2007) Mass spectrometry based proteomics in urine biomarker discovery. World Journal of Urology, 25, 435-443. doi:10.1007/s00345-007-0206-3
[10] Yamamoto, T., Langham, R.G., Ronco, P., Knepper, M.A. and Thongboonkerd, V. (2008) Towards standard protocols and guidelines for urine proteomics: A report on the human kidney and urine proteome project (HKUPP) symposium and workshop. Proteomics, 8, 2156-2159. doi:10.1002/pmic.200800138
[11] Bradford, M. A. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. doi:10.1016/0003-2697(76)90527-3
[12] Oh, J., Pyo, J.-H., Jo, E.-H., Hwang, S.-I., Kang, S.-C., Jung, J.-H., Park, E.-K., Kim, S.-Y., Choi, J.-Y. and Lim, J. (2004) Establishment of a near-standard two-dimensional human urine proteomic map. Proteomics, 4, 3485-3497. doi:10.1002/pmic.200401018
[13] Romani, A.M. and Scarpa, A. (2000) Regulation of cellular magnesium. Frontiers in Bioscience, 5, 720-734. doi:10.2741/Romani
[14] Ong, D.S., Mu, T.W., Palmer, A.E. and Kelly, J.W. (2010) Endoplasmic reticulum Ca2+ increases enhance mutant glucocerebrosidase proteostasis. Nature Chemical Biology, 6, 424-432. doi:10.1038/nchembio.368
[15] Verkoelen, C.F. and Verhulst, A. (2007) Proposed mechanisms in renal tubular crystal retention. Kidney International, 72, 13-18. doi:10.1038/
[16] Schlieper, G., Westenfeld, R., Brandenburg, V. and Ketteler, M. (2007) Inhibitors of calcification in blood and urine. Seminars in Dialysis, 20, 113-121. doi:10.1111/j.1525-139X.2007.00257.x
[17] De Yoreo, J.J., Qiu, S.R. and Hoyer, J.R. (2006) Molecular modulation of calcium oxalate crystallization. American Journal of Physiology Renal Physiology, 291, 11231131. doi:10.1152/ajprenal.00136.2006
[18] Held, I. R. and Freeman, S. (1964) Binding of calcium by human plasma proteins under simulated physiologic conditions. Journal of Applied Physiology, 19, 292-296.
[19] Berkelman T. (2008) Removal of interfering substances in samples prepared for two-dimensional (2-D) electrophoresis. Methods in Molecular Biology, 424, 51-62. doi:10.1007/978-1-60327-064-9_5
[20] Candiano, G., Santucci, L., Bruschi, M., Petretto, A., Ambrosio, C.D., Scaloni, A., Righetti, P.G. and Ghiggeri, G.M. (2012) “Cheek-to-cheek” urinary proteome profiling via combinatorial peptide ligand libraries: A novel, unexpected elution system. Journal of Proteomics, 75, 796-805. doi:10.1016/j.jprot.2011.09.018
[21] Adachi, J., Kumar, C., Zhang, Y., Olsen, J.V. and Mann, M. (2006) The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. Genome Biology, 7, R80.1-R80.16.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.