Sample preparation of gel electrophoretically separated protein binding partners for analysis by mass spectrometry.

The identification and characterization of binding partners from protein complexes is increasingly undertaken by mass spectrometry because of its high sensitivity and expedient elucidation of protein structure by accurate mass measurement. A variety of affinity purification methods including immunoprecipitation and glutathione-S-transferase (GST) pull-downs are commonly employed for the isolation of protein complexes and coupled to gel electrophoresis for further separation and basic information with regard to their constituents. For the successful analysis of gel-separated proteins by mass spectrometry, additional sample preparation steps involving sample clean-up, proteolysis, and peptide recovery are essential.

Two-dimensional gel electrophoresis for analysis of protein complexes.

Advances in genomic and proteomic technologies combined with molecular and cell biology have together enabled the identification of numerous genes and their products. Two-dimensional gel electrophoresis (2DE) is especially useful in the study of protein-protein interactions as it permits an improved separation of proteins as well as the detection of specific interacting protein isoform(s) of a protein resulting from post-translational modification. The investigation of interacting proteins using 2DE can be complemented by identification of the proteins by mass spectrometry.