High-Throughput Assessment of Kinome-wide Activation States.
Cell Syst
; 9(4): 366-374.e5, 2019 10 23.
Article
em En
| MEDLINE
| ID: mdl-31521607
ABSTRACT
Aberrant kinase activity has been linked to a variety of disorders; however, methods to probe kinase activation states in cells have been lacking. Until now, kinase activity has mainly been deduced from either protein expression or substrate phosphorylation levels. Here, we describe a strategy to directly infer kinase activation through targeted quantification of T-loop phosphorylation, which serves as a critical activation switch in a majority of protein kinases. Combining selective phosphopeptide enrichment with robust targeted mass spectrometry, we provide highly specific assays for 248 peptides, covering 221 phosphosites in the T-loop region of 178 human kinases. Using these assays, we monitored the activation of 63 kinases through 73 T-loop phosphosites across different cell types, primary cells, and patient-derived tissue material. The sensitivity of our assays is highlighted by the reproducible detection of TNF-α-induced RIPK1 activation and the detection of 46 T-loop phosphorylation sites from a breast tumor needle biopsy.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Neoplasias da Mama
/
Proteômica
/
Proteína Serina-Treonina Quinases de Interação com Receptores
/
Ensaios de Triagem em Larga Escala
Tipo de estudo:
Diagnostic_studies
Limite:
Female
/
Humans
Idioma:
En
Revista:
Cell Syst
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Holanda