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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)

Summary

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 

Abbreviations:

ACT

Acetone

CE

Capillary electrophoresis

CHAPS

[3-[(3-cholamidopropyl)dimethylammonio-1-propanesulfonate]

CHCA

α-Cyano-4-hydroxycinnamic acid

d

Dalton

2DE

Two-dimensional gel electrophoresis

DHB

Dihydroxybenzoic acid

DTE

1,4-Dithioerythritol

IEF

Isoelectric focusing

IPG

Immobilized pH gradient

LC

Liquid chromatography

MALDI

Matrix-assisted laser desorption ionization

MS

Mass spectrometry

MW

Molecular weight

MWCO

Molecular weight cut-off

ns

Nano-second

o/n

Overnight

RCF

Relative centrifugal forces

SA

Sinapinic acid

SDS

Sodium dodecylsulfate

SELDI

Surface-enhanced laser desorption

SPE

Solid phase extraction

TCA

Trichloroacetic acid

TFA

Trifluoroacetic acid

TGS

Tris-Glycine-SDS

TOF

Time of flight

UF

Ultrafiltration

Notes

Acknowledgments

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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