Ultrasensitive, multiplexed chemoproteomic profiling with soluble activity-dependent proximity ligation.
Proc Natl Acad Sci U S A
; 116(43): 21493-21500, 2019 10 22.
Article
em En
| MEDLINE
| ID: mdl-31591248
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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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cromatografia Líquida
/
Proteoma
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Proteômica
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Enzimas
/
Espectrometria de Massas em Tandem
/
Reação em Cadeia da Polimerase em Tempo Real
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Limite:
Humans
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Ano de publicação:
2019
Tipo de documento:
Article