Quantitative validation of different protein precipitation methods in proteome analysis of blood platelets

For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein comp...

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Bibliographic Details
Published in:Electrophoresis 2005-06, Vol.26 (12), p.2481-2489
Main Authors: Zellner, Maria, Winkler, Wolfgang, Hayden, Hubert, Diestinger, Michael, Eliasen, Maja, Gesslbauer, Bernd, Miller, Ingrid, Chang, Martina, Kungl, Andreas, Roth, Erich, Oehler, Rudolf
Format: Article
Language:English
Subjects:
Adult
Aged
Blood Platelets - chemistry
Blood Proteins - chemistry
Blood Proteins - drug effects
Blood Proteins - isolation & purification
Chemical Fractionation
Chemical Precipitation
Chromatography, High Pressure Liquid
Dialysis
Electrophoresis, Gel, Two-Dimensional - methods
Ethanol
Ethanol - pharmacology
Female
Humans
Male
Middle Aged
Nanotechnology
Platelets
Protein precipitation
Proteomics
Proteomics - methods
Silver Nitrate
Staining and Labeling
Trichloroacetic acid
Trichloroacetic Acid - pharmacology
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Summary:For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein composition of human blood platelet extracts after precipitation with ethanol (EtOH) or trichloroacetic acid (TCA). Both methods yielded the same amount of proteins from the platelet preparations. However, the EtOH‐precipitated samples had to be dialyzed because of the considerable salt content. To characterize single platelet proteins, samples were analyzed by two‐dimensional fluorescence differential gel electrophoresis. More than 90% of all the spots were equally present in the EtOH‐ and TCA‐precipitated samples. However, both precipitation methods showed a smaller correlation with nonprecipitated samples (EtOH 74.9%, TCA 79.2%). Several proteins were either reduced or relatively enriched in the precipitated samples. The proteins varied randomly in molecular weight and isoelectric point. This study shows that protein precipitation leads to specific changes in the protein composition of proteomics samples. This depends more on the specific structure of the protein than on the precipitating agent used in the experiment.
ISSN:0173-0835
1522-2683
DOI:10.1002/elps.200410262