Multiplex interrogration of protein-protein interactions in membrane-expressed multiprotein complexes
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[EMBARGOED UNTIL 12/01/2026] Intricate protein-protein interaction (PPI) networks coordinate cellular functions and govern the specificity of signal transduction. Distinct PPI network signatures can discern pathological states from healthy conditions, yet extracting this information from small clinical samples or rare cell populations remains technically challenging. Conventional PPI methods are often limited in throughput, typically interrogating one interaction at a time, require large sample volumes, extensive purification, or the introduction of tags and labels that can perturb native interactions. They are also technically complex, demanding specialized equipment and expertise, which limits adoptability and broad application. These constraints highlight the need for sensitive, multiplexed, and accessible platforms capable of simultaneously measuring multiple PPIs in physiologically relevant contexts. Here, we report the development and application of multiplexed immunoprecipitation detected by electrochemiluminescence (MIP-ECL), a plate-based ELISA procedure designed for ultrasensitive detection of PPIs from minimal biosamples using a Mesoscale QuickPlex platform. We focused on the T cell antigen receptor (TCR)/CD3 due to its central role in immunological tolerance and immunity. Technical development included validation of capture antibodies, assessment of capture affinity, and optimization of multiplex assay parameters. MIP-ECL assay optimization enabled remarkable sensitivity, allowing the detection of zepto-moles of native multiprotein complexes from physiological sources, and demonstrated that capture reagents spanning a range of affinities could be employed, with greatest sensitivity correlating with highest affinity. Using MIP-ECL, TCR/CD3 complexes marking leukemia in a mouse transplant model were detected in blood from pre-symptomatic mice as early as three days post-tumor injection. In the CD3δ knock-out mouse model of immunodeficiency, MIP-ECL revealed how subunit loss was accommodated within rare and poorly functional peripheral T cells. Furthermore, MIP-ECL uncovered a previously unrecognized co receptor (CD4:CD8α) co-association. This observation provides preliminary evidence of novel immunological interactions and illustrates MIP-ECL's potential as a PPI discovery tool. Taken together, this work establishes MIP-ECL as a versatile, ultrasensitive, and adoptable platform for the validation, hypothesis-driven experimentation and discovery-driven mapping of PPIs within multiprotein complexes. By enabling analysis of rare cells and small physiological samples, MIP-ECL provides new opportunities to explore PPI networks, uncover previously unappreciated interactions, and advance our understanding of PPI in health and disease. We propose that our assay will permit detailed network analysis that has the potential to reveal context-dependent modulations in multiprotein signaling, providing insights that could significantly inform and refine pharmacological intervention strategies
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Ph. D.