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. This chapter describes the important procedure of in-gel digestion with particular emphasis on maximum peptide recovery and compatibility for subsequent mass spectrometric analysis.