Summary
Reproducible procedures for the preparation of protein samples isolated from human urine are essential for meaningful proteomic analyses. Key applications are the discovery of novel proteins or their modifications in the human urine as well as protein biomarker discovery for diseases and drug treatments. The methodology presented here features experimental steps aimed at limiting protein losses because of organic solvent precipitation, effective separation of proteins from other compounds in the human urine and molecular weight-based enrichment of proteins in two distinct fractions. Urinary proteins are separated from cellular debris in the urine via centrifugation, concentrated with 5-kDa-cutoff membrane concentration devices and separated via size exclusion chromatography into fractions with a higher and a lower molecular weight than 30 kDa, respectively. A successive optional affinity removal step for highly abundant plasma proteins is described. Finally, buffer exchange steps useful for specific downstream proteomic analysis experiments of urinary proteins are presented, such as 2-dimensional gel electrophoresis, differential protein or peptide labeling and digestion with trypsin for LC-MS/MS analysis.
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Pieper, R. (2008). Preparation of Urine Samples for Proteomic Analysis. In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. Methods in Molecular Biology™, vol 425. Humana Press. https://doi.org/10.1007/978-1-60327-210-0_8
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DOI: https://doi.org/10.1007/978-1-60327-210-0_8
Publisher Name: Humana Press
Print ISBN: 978-1-60327-209-4
Online ISBN: 978-1-60327-210-0
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