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 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.
Zellner
Maria
M
Surgical Research Laboratories, Medical University of Vienna, Vienna, Austria.
Winkler
Wolfgang
W
Hayden
Hubert
H
Diestinger
Michael
M
Eliasen
Maja
M
Gesslbauer
Bernd
B
Miller
Ingrid
I
Chang
Martina
M
Kungl
Andreas
A
Roth
Erich
E
Oehler
Rudolf
R
D000328:Adult; D000368:Aged; D001792:Blood Platelets / Q000737:chemistry*; D001798:Blood Proteins / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification*; D005591:Chemical Fractionation; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D003956:Dialysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D000431:Ethanol / Q000494:pharmacology; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D036103:Nanotechnology; D040901:Proteomics / Q000379:methods*; D012835:Silver Nitrate; D013194:Staining and Labeling; D014238:Trichloroacetic Acid / Q000494:pharmacology
D001798:Blood Proteins; D000431:Ethanol; D014238:Trichloroacetic Acid; D012835:Silver Nitrate