ABSTRACT: This section describes the biological relevance, intent, and overview of the ultra-fast quantitative proteomics coupled with immunoprecipitation (IP) experiment, which specifically captures tagged proteins from the EV_IP-Flag control, wild-type (WT) BTK, and a comprehensive panel of BTK single and combinatorial mutants. 1. Biological Relevance & Experimental Intent Bruton’s tyrosine kinase (BTK) is a central regulator of B-cell receptor (BCR) signaling, with dysregulated activity driving the pathogenesis of B-cell malignancies and autoimmune diseases. While the kinase domain of BTK is well-characterized, the impact of clinically relevant mutations—including single, double, triple, and quadruple variants of Cys481, Leu408, Arg525, and Gln412—on its interactome and downstream signaling remains incompletely defined. These mutations are frequently associated with acquired resistance to BTK inhibitors (BTKi), yet their combinatorial effects on protein-protein interactions (PPIs) and signaling rewiring are poorly understood. This experiment aims to systematically compare the interactomes of WT BTK and an extended panel of BTK mutants, with the goal of identifying mutation-specific binding partners that drive resistance or altered signaling. By profiling the interactome of each variant against an empty vector (EV_IP-Flag) control, we aim to uncover: Core BTK interactors conserved across all variants Single-mutation-specific interactors that may mediate drug resistance Combinatorial mutation-induced interactome rewiring that enhances or alters signaling Signaling complexes underlying functional differences between WT and mutant BTK 2. Experimental Overview We employed ultra-fast quantitative proteomics combined with anti-Flag immunoprecipitation to profile the interactomes of 16 experimental groups: EV_IP-Flag (empty vector negative control, expressing only the Flag tag) WT BTK (wild-type BTK-Flag) BTK GLN412→GLY (kinase domain mutant) BTK ARG525→GLY (kinase domain mutant) BTK LEU408→ARG (kinase domain mutant) BTK CYS481→ARG (BTKi-resistant mutant) BTK CYS481→ARG + ARG525→GLY (double mutant) BTK CYS481→ARG + LEU408→ARG (double mutant) BTK CYS481→ARG + GLN412→GLY (double mutant) BTK ARG525→GLY + LEU408→ARG (double mutant) BTK ARG525→GLY + GLN412→GLY (double mutant) BTK LEU408→ARG + GLN412→GLY (double mutant) BTK CYS481→ARG + ARG525→GLY + LEU408→ARG (triple mutant) BTK CYS481→ARG + ARG525→GLY + GLN412→GLY (triple mutant) BTK CYS481→ARG + LEU408→ARG + GLN412→GLY (triple mutant) BTK ARG525→GLY + LEU408→ARG + GLN412→GLY (triple mutant) BTK CYS481→ARG + ARG525→GLY + LEU408→ARG + GLN412→GLY (quadruple mutant) Briefly, cells were transfected with the respective constructs, and whole-cell lysates were subjected to immunoprecipitation using an anti-Flag antibody to enrich Flag-tagged BTK (or the tag alone in the EV control) and its associated proteins. Captured protein complexes were digested, and peptides were analyzed by ultra-fast LC-MS/MS, enabling high-throughput identification and quantification of interactors across all samples. The EV_IP-Flag control was used to subtract non-specific interactors and background binding, ensuring only specific BTK-associated proteins were retained for downstream analysis. Differential abundance analysis was performed to compare interactors between WT BTK and each mutant, as well as against the EV control, to identify proteins significantly enriched or depleted in each variant. These results provide a comprehensive map of BTK interactome changes induced by clinically relevant single and combinatorial mutations, offering mechanistic insights into BTKi resistance and identifying potential novel therapeutic targets.
Xi Chen 
