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

Edited by James A. Wells, University of California, San Francisco, CA, and approved September 12, 2019 (received for review July 26, 2019)
October 7, 2019
116 (43) 21493-21500

Significance

We report the development of a chemical proteomic platform, soluble activity-dependent proximity ligation (sADPL), which enables ultrasensitive and multiplexed quantification of endogenous active proteins in complex proteome samples from cells, fluids, and tissues. Single-plexed and multiplexed sADPL can be implemented to “write” and “read” barcoded oligonucleotide amplicons derived from specific active enzymes in extremely low levels of whole proteome. We apply sADPL to quantify in vivo protein–drug interactions from blood samples, as well as perform hundreds to thousands of parallel activity measurements in fresh or flash-frozen patient tumor samples in a matter of hours on a benchtop. Combined, these studies provide compelling proof-of-concept examples for future applications for the molecular analysis of biological and clinical samples.

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.

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Acknowledgments

We thank all the patients who kindly donated samples. We thank C. He for access to instrumentation, P. Dauer for assistance with mouse PBMC isolation, and S. Ahmadiantehrani for proofreading assistance. This work was supported by NIH Chemical Biology Interface Training Grant 2T32GM008720-16 ( to J.E.M.), the Marsha Rivkin Foundation (M.A.E.), National Cancer Institute Grant R01 CA111882 (to E.L.), NIH Grants R00 CA175399 and DP2 GM128199-01 (to R.E.M.), the Chicago Biomedical Consortium supported by Searle Family Funds (R.E.M.), University of Chicago Cancer Center Support Grant P30 CA014599, and the Duchossois Family Institute at the University of Chicago.

Supporting Information

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Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 116 | No. 43
October 22, 2019
PubMed: 31591248

Classifications

Submission history

Published online: October 7, 2019
Published in issue: October 22, 2019

Keywords

  1. chemoproteomics
  2. chemical probes
  3. proteomics
  4. diagnostics
  5. proximity ligation

Acknowledgments

We thank all the patients who kindly donated samples. We thank C. He for access to instrumentation, P. Dauer for assistance with mouse PBMC isolation, and S. Ahmadiantehrani for proofreading assistance. This work was supported by NIH Chemical Biology Interface Training Grant 2T32GM008720-16 ( to J.E.M.), the Marsha Rivkin Foundation (M.A.E.), National Cancer Institute Grant R01 CA111882 (to E.L.), NIH Grants R00 CA175399 and DP2 GM128199-01 (to R.E.M.), the Chicago Biomedical Consortium supported by Searle Family Funds (R.E.M.), University of Chicago Cancer Center Support Grant P30 CA014599, and the Duchossois Family Institute at the University of Chicago.

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

Gang Li
Department of Chemistry, The University of Chicago, Chicago, IL 60637;
Institute for Genomics and Systems Biology, The University of Chicago, Chicago, IL 60637;
Mark A. Eckert
Section of Gynecologic Oncology, Department of Obstetrics and Gynecology, The University of Chicago, Chicago, IL 60637
Jae Won Chang
Department of Chemistry, The University of Chicago, Chicago, IL 60637;
Institute for Genomics and Systems Biology, The University of Chicago, Chicago, IL 60637;
Jeffrey E. Montgomery
Department of Chemistry, The University of Chicago, Chicago, IL 60637;
Institute for Genomics and Systems Biology, The University of Chicago, Chicago, IL 60637;
Agnieszka Chryplewicz
Section of Gynecologic Oncology, Department of Obstetrics and Gynecology, The University of Chicago, Chicago, IL 60637
Ernst Lengyel
Section of Gynecologic Oncology, Department of Obstetrics and Gynecology, The University of Chicago, Chicago, IL 60637
Raymond E. Moellering1 [email protected]
Department of Chemistry, The University of Chicago, Chicago, IL 60637;
Institute for Genomics and Systems Biology, The University of Chicago, Chicago, IL 60637;

Notes

1
To whom correspondence may be addressed. Email: [email protected].
Author contributions: G.L., M.A.E., J.W.C., E.L., and R.E.M. designed research; G.L., M.A.E., J.W.C., J.E.M., and A.C. performed research; G.L., M.A.E., J.W.C., E.L., and R.E.M. contributed new reagents/analytic tools; G.L., M.A.E., J.W.C., E.L., and R.E.M. analyzed data; and G.L. and R.E.M. wrote the paper.

Competing Interests

The authors declare no competing interest.

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    Ultrasensitive, multiplexed chemoproteomic profiling with soluble activity-dependent proximity ligation
    Proceedings of the National Academy of Sciences
    • Vol. 116
    • No. 43
    • pp. 21333-21950

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