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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

  • Protocol
Clinical Proteomics

Part of the book series: Methods in Molecular Biology™ ((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.

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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

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Acknowledgments

This study was supported by the Greek Ministry of Health.

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Authors and Affiliations

  1. Division of Biotechnology, Biomedical Research Foundation, Academy of Athens, Athens, Greece

    Panagiotis G. Zerefos

  2. Division of Biotechnology, Biomedical Research Foundation, Academy of Athens, Athens, Greece

    Antonia Vlahou

Authors
  1. Panagiotis G. Zerefos
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  2. Antonia Vlahou
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Editor information

Editors and Affiliations

  1. Biomedical Research Foundation, Academy of Athens, Athens, Greece

    Antonia Vlahou

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

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Zerefos, P.G., Vlahou, A. (2008). Urine Sample Preparation and Protein Profiling by Two-Dimensional Electrophoresis and Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectroscopy. In: Vlahou, A. (eds) Clinical Proteomics. Methods in Molecular Biology™, vol 428. Humana Press. https://doi.org/10.1007/978-1-59745-117-8_8

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  • DOI: https://doi.org/10.1007/978-1-59745-117-8_8

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-837-9

  • Online ISBN: 978-1-59745-117-8

  • eBook Packages: Springer Protocols

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