Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography.
We describe an approach for fractionating complex protein samples prior to two-dimensional gel electrophoresis using reversed-phase high-performance liquid chromatography. Whole lysates of cells and tissue were prefractionated by reversed-phase chromatography and elution with a five-step gradient of increasing acetonitrile concentrations. The proteins obtained at each step were subsequently separated by high-resolution two-dimensional gel electrophoresis (2-DE). The reproducibility of this prefractionation technique proved to be optimal for comparing 2-DE gels from two different cell states. In addition, this method is suitable for enriching low-abundance proteins barely detectable by silver staining to amounts that can be detected by Coomassie blue and further analyzed by mass spectrometry.
Badock
V
V
Max-Delbruck-Center for Molecular Medicine, Department of Protein Chemistry, Berlin, Germany.
Steinhusen
U
U
Bommert
K
K
Otto
A
A
D000818:Animals; D017209:Apoptosis; D001402:B-Lymphocytes / Q000737:chemistry; D001940:Breast / Q000737:chemistry; D002460:Cell Line / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004396:Coloring Agents; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004847:Epithelial Cells / Q000737:chemistry; D005260:Female; D006801:Humans; D008168:Lung / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D020543:Proteome; D051381:Rats; D015203:Reproducibility of Results; D012394:Rosaniline Dyes; D016622:Silver Staining; D013048:Specimen Handling / Q000379:methods; D013194:Staining and Labeling; D013382:Subtraction Technique
D004396:Coloring Agents; D011506:Proteins; D020543:Proteome; D012394:Rosaniline Dyes; C048139:Coomassie blue