Ultrasensitive, multiplexed chemoproteomic profiling with soluble activity-dependent proximity ligation

Abstract

Chemoproteomic methods can report directly on endogenous, active enzyme populations, which can differ greatly from measures of transcripts or protein abundance alone. Detection and quantification of family-wide probe engagement generally requires LC-MS/MS or gel-based detection methods, which suffer from low resolution, significant input proteome requirements, laborious sample preparation, and expensive equipment. Therefore, methods that can capitalize on the broad target profiling capacity of family-wide chemical probes but that enable specific, rapid, and ultrasensitive quantitation of protein activity in native samples would be useful for basic, translational, and clinical proteomic applications. Here we develop and apply a method that we call soluble activity-dependent proximity ligation (sADPL), which harnesses family-wide chemical probes to convert active enzyme levels into amplifiable barcoded oligonucleotide signals. We demonstrate that sADPL coupled to quantitative PCR signal detection enables multiplexed “writing” and “reading” of active enzyme levels across multiple protein families directly at picogram levels of whole, unfractionated proteome. sADPL profiling in a competitive format allows for highly sensitive detection of drug–protein interaction profiling, which allows for direct quantitative measurements of in vitro and in vivo on- and off-target drug engagement. Finally, we demonstrate that comparative sADPL profiling can be applied for high-throughput molecular phenotyping of primary human tumor samples, leading to the discovery of new connections between metabolic and proteolytic enzyme activity in specific tumor compartments and patient outcomes. We expect that this modular and multiplexed chemoproteomic platform will be a general approach for drug target engagement, as well as comparative enzyme activity profiling for basic and clinical applications.

In Vivo Imaging of the Tumor‐Associated Enzyme NCEH1 with a Covalent PET Probe

“…Gel‐based measurements of active enzyme in microdissected tumor cells from the edge and core of xenografts were made using a specific fluorescent probe that labels active NCEH1, JW576, which confirmed significantly higher NCEH1 activity in cells isolated from the edge of both MDA‐MB231 and PC3 xenografts (Figure B and Figures S7 and S8 C). This difference in active NCEH1 was also confirmed using a more sensitive method, soluble activity‐dependent proximity ligation (Figure C) . Likewise, western blot revealed decreased NCEH1 abundance in the tumor core relative to the edge (Figure D).…”

Site-specific DNA functionalization through the tetrazene-forming reaction in ionic liquids

“…13,14 The methods of chemical synthesis of DNA have signicantly facilitated the introduction of desired functionalities onto a specic position, 15 and post-synthetic modication methods are also powerful chemical tools, [16][17][18][19] especially for labeling of DNA targets with large labeling groups incompatible during the chemical synthesis process. [20][21][22] Bioinspired enzymatic postsynthetic labeling technologies have emerged as useful approaches through sequence-or structure-dependent substrate recognition, 23 and chemical modication is an alternative means for site-specic DNA labeling by incorporation of a chemical handle that is inert toward endogenous chemical functional groups but reactive toward a labeling reagent. 24,25 However, despite the remarkable advance of the bioconjugation technologies during the past decades, site-specic chemical modication remains a serious challenge due to its demanding criteria which include adequate reactivity of the labeling reagents toward the target site and high selectivity in biomolecule-compatible solvents such as aqueous buffer.…”

In Vivo Imaging of the Tumor‐Associated Enzyme NCEH1 with a Covalent PET Probe

“…This difference in active NCEH1 was also confirmed using a more sensitive method, soluble activity-dependent proximity ligation (Figure 3 C). [24,25] Likewise, western blot revealed decreased NCEH1 abundance in the tumor core relative to the edge (Figure 3 D). Finally, immunofluorescent imaging of whole xenograft tissue sections confirmed NCEH1 localization along the outer edges of the tumor, with comparatively little NCEH1 observed within the tumor, reinforcing the microenvironmental heterogeneity observed by PET imaging with [ 18 F]JW199 (Figure 3 E).…”