Profiling of BCLxL Protein Complexes in Non-Small Cell Lung Cancer Cells via Multiplexed Single-Molecule Pull-Down and Co-Immunoprecipitation.

We introduce multiplexed single-molecule pull-down and co-immunoprecipitation, named m-SMPC, an analysis tool for profiling multiple protein complexes within a single reaction chamber using single-molecule fluorescence imaging. We employed site-selective conjugation of biotin and fluorescent dye directly onto the monoclonal antibodies, which completed an independent sandwich immunoassay without the issue of host cross-reactivity. We applied this technique to profile endogenous B-cell lymphoma extra-large (BCLxL) complexes in non-small cell lung cancer (NSCLC) cells. Up to three distinct BCLxL complexes were successfully detected simultaneously within a single reaction chamber without fluorescence signal crosstalk. Notably, the NSCLC cell line EBC-1 exhibited high BCLxL-BAX and BCLxL-BAK levels, which closely paralleled a strong response to the BCLxL inhibitor A-1331852. This streamlined method offers the potential for quantitative biomarkers derived from protein complex profiling, paving the way for their application in protein complex-targeted therapies.