An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells

Li, Gang; Montgomery, Jeffrey E.; Eckert, Mark A.; Chang, Jae Won; Tienda, Samantha M.; Lengyel, Ernst; Moellering, Raymond E.

doi:10.1038/s41467-017-01854-0

Cited by 39 publications

(38 citation statements)

References 51 publications

(63 reference statements)

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“…sADPL integrates the activity-dependent and family-wide tagging of endogenous, active enzymes using chemical probes with the specific and robust signal amplification afforded by barcoded oligonucleotide proximity ligation and amplification ( Fig. 1) (15)(16)(17)(18). In contrast to the majority of studies that only use chemical probes in homogenized cell lysates, we sought to label active enzymes in their native environment, and therefore performed sADPL by pulsing live cells with one probe or a combination of several family-wide probes (Fig.…”

Section: Results Sadpl Enables Ultrasensitive and Specific Activity Mmentioning

confidence: 99%

“…We recently reported an imaging-based activity-dependent proximity ligation platform, termed ADPL imaging, which enables spatially resolved detection of active enzymes in single cells using family-wide probes (15). While the concept behind this approach addresses several shortcomings, the imaging-based format places limitations on samples types that can be interrogated (i.e., incompatible with biofluids), as well as low processing throughput involved with sample preparation and microscopy.…”

Section: Significancementioning

confidence: 99%

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

Eckert

Chang

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Results Sadpl Enables Ultrasensitive and Specific Activity Mmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

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

Eckert

Chang

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…Coincidence of the probe‐ and protein‐directed dual antibody complexes enables specific ligation of a bridging oligonucleotide and rolling circle amplification. Finally, the ADPL signal is detected by means of fluorescence microscopy after incubation with a complementary, fluorophore‐labeled oligonucleotide . We speculated that a potential liability of the dual primary–secondary antibody sandwich complex, which could theoretically extend up to about 40 nm from either the probe or POI, might result in a background signal being generated from complexes formed on the POI and endogenous biotinylated proteins or other non‐POI targets of the probe.…”

Section: Resultsmentioning

confidence: 99%

A Concise, Modular Antibody–Oligonucleotide Conjugation Strategy Based on Disuccinimidyl Ester Activation Chemistry

Moellering

2019

ChemBioChem

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The synthesis of antibody–oligonucleotide conjugates has enabled the development of highly sensitive bioassays for specific epitopes in the laboratory and clinic. Most synthetic schemes to generate these hybrid molecules require expensive reagents, significant quantities of input antibody, and multistep purification routes; thus limiting widespread application. Herein a facile and robust conjugation strategy is reported that involves “plug‐and‐play” antibody conjugation with succinimidyl‐functionalized oligonucleotides, which are high yielding and compatible for use directly after buffer exchange. The succinimidyl‐linked oligonucleotides are synthesized with 5′‐amine‐modified oligonucleotides and disuccinimidyl suberate (DSS), both of which are inexpensive and commercially available. Direct incubation of the resulting stable succinimidyl– oligonucleotide conjugates with commercial antibodies yields conjugates ready for use after benchtop buffer exchange. It is demonstrated that the resulting oligonucleotide–antibody and oligonucleotide–streptavidin conjugates retain potent and specific binding in activity‐dependent proximity ligation imaging, and proximity ligation‐mediated qPCR detection of endogenous proteins in native cellular contexts down to picogram levels of whole proteome. This DSS conjugation strategy should be widely applicable in the synthesis of protein–oligonucleotide conjugates.

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“…1c). [18][19][20] Innovative recent examples of PLA assays applied to chemical biology include ultrasensitive detection of posttranslational modifications 19,21 (e.g., Glycoseek 22 ) and antibodies, 23,24 as well as assessing hydrolytic 20 and kinase 25 enzyme activity (e.g., activity-dependent proximity ligation, ADPL).…”

Section: Resultsmentioning

confidence: 99%

An Activity-Based Methionine Bioconjugation Approach to Developing Proximity-Activated Imaging Reporters

Ohata¹,

Krishnamoorthy²,

González³

et al. 2019

Preprint

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Chemical probes that report on protein activity, rather than protein abundance, with spatial and temporal resolution can enable studies of their native function in biological contexts as well as provide opportunities for developing new types of biochemical reporters. Here we present a sensing platform, termed proximity-activated imaging reporter (PAIR), which combines activity-based methionine bioconjugation and antibody labeling with proximity-dependent oligonucleotide-based amplification to monitor dynamic changes of a given analyte in cells and animals through context-dependent methionine labeling of specific protein targets. We establish this PAIR method to develop sensors for imaging reactive oxygen species (ROS) and calcium ions through oxaziridine-directed labeling of reactive methionine residues on β-actin and calmodulin (CaM), respectively, where the extent of methionine bioconjugation on these protein targets can serve as an indicator of oxidative stress or calcium status. In particular, application of PAIR to activity-based CaM detection provides a method for imaging integrated calcium activity in both in vitro cell and in vivo zebrafish models. By relying on native protein biochemistry, PAIR enables redox and metal imaging without introduction of external small-molecules or genetically encoded indicators that can potentially buffer the natural/existing pools. This approach can be potentially generalized to target a broader range of analytes by pairing appropriate activity-based protein probes with protein detection reagents in a proximity-driven manner, providing a starting point not only for designing new sensors but also for monitoring endogenous activity of specific protein targets in biological specimens with spatial and temporal fidelity.

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An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells

Cited by 39 publications

References 51 publications

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

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

A Concise, Modular Antibody–Oligonucleotide Conjugation Strategy Based on Disuccinimidyl Ester Activation Chemistry

An Activity-Based Methionine Bioconjugation Approach to Developing Proximity-Activated Imaging Reporters

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