Dynamic cis-proteomics comprehensively reveals signal-dependent regulators Xinying Zong1, Beisi Xu2, Vishwajeeth R. Pagala3, Minghong He1, Yong-Dong Wang4, Xiaolei Hao1, Lu Bai1, Jun Li1, Menglin Jiang1, Ji-Hoon Cho3, Richard Cross1, Jon P. Connelly5, Shondra M. Pruett-Miller5, Junmin Peng3,6, Yongqiang Feng1,7* 1. Department of Immunology 2. Center for Applied Bioinformatics 3. Center for Proteomics and Metabolomics 4. Department of Cell and Molecular Biology 5. Center for Advanced Genome Engineering 6. Department of Structure Biology and Department of Developmental Neurobiology St. Jude Children’s Research Hospital 7. Lead contact Funding: American Lebanese Syrian Associated Charities (Y.F.) and NIH grants R21AI146614 (Y.F.) and R01AG053987 (J.P.). Regulation of gene expression by environmental cues governs cell differentiation and function. Here, we developed a comparative proteomics approach to profile the dynamic protein components at genetic elements of interest (cis-proteome) in situ in response to stimulation. Our antibody-mediated proximity biotinylation with tunable spatial resolution enables the capturing of cis-proteome in situ without genetic engineering but with high sensitivity and robustness, allowing the comparative analysis of the dynamics of cis-proteome in different contexts. Applying it to mouse regulatory T cells comprehensively unveiled the proteins involved in Interleukin-2 or T cell antigen receptor signaling–dependent gene regulation, highlighting the broad applicability and power of profiling dynamic cis-proteome.