Identification of Akt‐interacting proteins using on‐beads cross‐linking, co‐immunoprecipitation and mass spectrometry

Akt is a critical signaling molecule that regulates cell survival. However, detailed signaling mechanisms of Akt are not fully understood, in part because its interacting proteins are not well‐defined. In the study, we developed a novel approach for identifying binding partners of Akt in Neuro 2A cells. We used a two‐step cross‐linking strategy that overcomes common problems in co‐immunoprecipitation (IP)/MS experiments such as antibody contamination or the loss of interacting partners during washing, particularly for the weakly associating interactors. Akt antibody was immobilized on protein beads by cross‐linking with non‐cleavable succinimidyl suberate (DSS) to avoid antibody elution. Subsequent treatment of the immobilized Akt‐antibody with cell lysates followed by a cleavable crosslinker, dithiobis[succinimidylpropionate] (DSP), produced stable complexes between Akt and its binding partners. After extensive washing, the co‐IP products were eluted with Laemmli buffer containing 5% β‐mercaptoethanol that cleaves DSP cross‐linking. The liberated proteins were subjected to SDS‐PAGE/in‐gel tryptic digestion/nanoLC‐MS/MS analysis. This approach allowed us to identify several new proteins as potential Akt interacting partners, in addition to seven previously reported Akt‐binding proteins, from cell lysates containing as low as 1.5 mg proteins.