Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes

Proteins often assemble into multiprotein complexes for carrying out their biological functions. Affinity purification combined with mass spectrometry (AP-MS) is a method of choice for unbiasedly charting protein complexes. Typically, genetically tagged bait protein and associated proteins are immunoprecipitated from cell lysate and subjected to in-gel or on-bead digestion for MS analysis. However, the sample preparation procedures are often time-consuming and skipping reduction and alkylation steps results in incomplete digestion. Here, by seamlessly combining AP with the simple and integrated spintip-based proteomics technology (SISPROT), we developed an integrated AP-MS workflow for simultaneously processing more than 10 AP samples from cells cultured in six-well plates in 2 h. Moreover, we developed a quantitation-based data analysis workflow for differentiating potential interacting proteins from nonspecific interferences. The AP-SISPROT ensures high digestion efficiency especially for large transmem...