Reducing sample complexity by RP-HPLC: beyond the tip of the protein expression iceberg.
Because the dynamic range of most cell or tissue proteomes is enormous, separation of such complex protein samples by two-dimensional electrophoresis (2-DE) on broad pH gradients often results in the visualization of only the most abundantly expressed proteins. It is, therefore, often beneficial to first subdivide the proteome in smaller, less complex fractions before 2-DE. This enables the analysis of a larger number of proteins. One approach to prefractionate protein samples is by reversed-phase high-performance liquid chromatography (RP-HPLC), separating proteins according to their hydrophobicity. This effectively introduces a third separation dimension, increasing the spatial resolution of the experiment. Here, we will describe a procedure for separating whole protein lysates by RP-HPLC, before their analysis by 2-DE or 2-D difference gel electrophoresis.
Van den Bergh
Gert
G
Katholieke Universiteit Leuven, Leuven, Belgium.
Arckens
Lutgarde
L
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D014176:Protein Biosynthesis / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D020543:Proteome / Q000032:analysis* / Q000378:metabolism
D011506:Proteins; D020543:Proteome