Urine Sample Preparation and Protein Profiling by Two-Dimensional Electrophoresis and Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectroscopy

  • Panagiotis G. Zerefos
  • Antonia Vlahou
Part of the Methods in Molecular Biology™ book series (MIMB, volume 428)


Urine represents the most easily attainable and consequently one of the most common samples in clinical analysis and diagnostics. However, urine is also considered one of the most difficult proteomic samples to work with due to its highly variable contents, as well as the presence of various proteins in low abundance or modified forms. In this chapter, we describe simple protocols and troubleshooting tips for urinary protein preparation and profiling by two-dimensional electrophoresis or directly via matrix-assisted laser desorption ionization time of flight mass spectroscopy. Direct dilution, protein precipitation, ultrafiltration, and solid phase extraction in combination to the above profiling technologies serve the means for reliable proteomics analysis of one of the most significant yet very complex biological samples.

Key Words

urine 2DE MALDI-TOF-MS protein profiling sample preparation 





Capillary electrophoresis




α-Cyano-4-hydroxycinnamic acid




Two-dimensional gel electrophoresis


Dihydroxybenzoic acid




Isoelectric focusing


Immobilized pH gradient


Liquid chromatography


Matrix-assisted laser desorption ionization


Mass spectrometry


Molecular weight


Molecular weight cut-off






Relative centrifugal forces


Sinapinic acid


Sodium dodecylsulfate


Surface-enhanced laser desorption


Solid phase extraction


Trichloroacetic acid


Trifluoroacetic acid




Time of flight





This study was supported by the Greek Ministry of Health.


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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Panagiotis G. Zerefos
    • 1
  • Antonia Vlahou
    • 2
  1. 1.Division of Biotechnology, Biomedical Research FoundationAcademy of AthensAthensGreece
  2. 2.Division of Biotechnology, Biomedical Research FoundationAcademy of AthensAthensGreece

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