Abstract
Urine has garnered tremendous interest over the past decade as a potential source of protein biomarkers for various pathologies. However, due to its low protein concentration and the presence of interfering compounds, urine constitutes a challenging analyte in proteomics. In the context of a project aimed at the discovery and evaluation of new candidate biomarkers of bladder cancer in urine, our laboratory has implemented and evaluated an array of preparation techniques for urinary proteome analysis. We present here the protocol that, in our hands, yielded the best overall proteome coverage with the lowest analytical effort. It begins with protein precipitation using trichloroacetic acid, in solution digestion and RP-C18 cartridge desalting of the resulting peptides mixture, and is followed by peptide fractionation by gel-free isoelectric focusing, and nano-LC-MS/MS for database compilation.
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Acknowledgements
Dr. Dimitri Vordos, Edwige Lopes, Yves Allory, Hopital Mondor in Paris, and Nuria Mallats CNIO in Madrid are gratefully acknowledged for their assistance in specimen collection. The authors thank Dr. Christophe Bruley and Veronique Dupierris, CEA Grenoble, for the IRMa software and his help with AMT tag database compilation, and Madalen LeGorrec, CEA Grenoble, for the protein dosage and the pools constitution. The authors thank Dr. Elodie Duriez from CRP-Santé in Luxembourg for her comments and helpful discussion. The authors also express gratitude to all the members of the DECanBio consortium for their help and advice. This work was funded through the EU FP7 program DECanBio (www.decanbio.eu) [Grant Agreement #201333]. The Decon-2LS and VIPER software used for data processing were provided by the W. R. Wiley Environmental Molecular Science Laboratory, a national scientific user facility sponsored by the US Department of Energy’s Office of Biological and Environmental Research, located at PNNL. PNNL is operated by Battelle Memorial Institute for the US Department of Energy under contract DE-AC05-76RL0 1830.
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Court, M., Garin, J., Masselon, C.D. (2015). Urine Sample Preparation and Fractionation for Global Proteome Profiling by LC-MS. In: Vlahou, A., Makridakis, M. (eds) Clinical Proteomics. Methods in Molecular Biology, vol 1243. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1872-0_10
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DOI: https://doi.org/10.1007/978-1-4939-1872-0_10
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