Overview: Co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS) Protein Profiling (ID) is a powerful technique used to identify protein-protein interactions and protein complexes. This technique involves the specific capture of proteins bound to a target protein using target protein-specific antibodies. The captured proteins are then identified by mass spectrometry analysis.
Immunoprecipitation: Start by lysing the cells of interest with a gentle lysis buffer that is suitable for maintaining protein interactions. Incubate the lysate with antibodies or other proteins that recognizes the protein of interest. After incubation, add beads to capture the antibody-protein or protein-protein complex. The beads can be washed several times with the lysis buffer to remove nonspecific binding. The protein complex bound to the beads can be eluted using a low pH buffer, such as glycine or citrate buffer, or urea solution.
Sample Preparation: After elution, the protein complex can be separated by SDS-PAGE and visualized by Coomassie or silver staining. The band of interest are excised and subjected to in-gel digestion using trypsin. The protein complex can also be cleaned by desalt cartridges before in-solution digestion.
High-performance liquid chromatography – mass spectrometry (HPLC-MS) analysis: The peptide mixture obtained from in-gel or in-solution digestion is separated using reversed phase (RP) high-performance liquid chromatography (HPLC). The peptides are ionized and analyzed using mass spectrometry to determine the mass-to-charge ratio of each parental and fragmental ions for proteins in the complex.
Data analysis: The MS data are analyzed using proteomics analysis software tools to identify the proteins present in the complex and their relative abundance.