Abstract 1142: An activity-dependent proximity ligation platform for spatially resolved and multiplexed quantifications of active enzymes in single cells

Integration of chemical probes into proteomic workflows enables the interrogation of protein activity, rather than abundance. Current methods limit the biological contexts that can be addressed due to sample homogenization, signal-averaging, and bias toward abundant proteins. To expand the application scope of chemical proteomics and explore the cellular heterogeneity on enzyme activities, we developed a new platform that integrates family-wide chemical probes with proximity-dependent oligonucleotide amplification and imaging to quantify enzyme activity in native contexts with high spatial and single cell resolution. Application of this method, activity-dependent proximity ligation (ADPL), to serine hydrolase and cysteine protease enzymes enables quantification of differential enzyme activity resulting from endogenous changes in localization and expression. In a competitive format, small molecule target engagement with endogenous proteins in live cells can be quantified. Retention of sample architecture enables interrogation of complex environments such as cellular co-culture and patient samples. We also successfully applied ADPL in xenograft tissue sample, exhibiting the application prospect in primary patient tissue. Additionally, implementation of barcoded antibody-oligo direct conjugation enabled multiplexed readout of active enzymes within and between enzyme families, providing the possibility of simultaneous biomarker detection. Together, this work supports ADPL should be amenable to diverse and multiplexed protein families to detect active enzymes at scale and resolution out of reach with current methods. Citation Format: Gang Li, Raymond E. Moellering. An activity-dependent proximity ligation platform for spatially resolved and multiplexed quantifications of active enzymes in single cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1142.