Affinity-based profiling of endogenous phosphoprotein phosphatases by mass spectrometry.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
10 2021
10 2021
Historique:
received:
08
02
2021
accepted:
12
07
2021
pubmed:
15
9
2021
medline:
18
11
2021
entrez:
14
9
2021
Statut:
ppublish
Résumé
Phosphoprotein phosphatases (PPPs) execute >90% of serine/threonine dephosphorylation in cells and tissues. While the role of PPPs in cell biology and diseases such as cancer, cardiac hypertrophy and Alzheimer's disease is well established, the molecular mechanisms governing and governed by PPPs still await discovery. Here we describe a chemical proteomic strategy, phosphatase inhibitor beads and mass spectrometry (PIB-MS), that enables the identification and quantification of PPPs and their posttranslational modifications in as little as 12 h. Using a specific but nonselective PPP inhibitor immobilized on beads, PIB-MS enables the efficient affinity-capture, identification and quantification of endogenous PPPs and associated proteins ('PPPome') from cells and tissues. PIB-MS captures functional, endogenous PPP subunit interactions and enables discovery of new binding partners. It performs PPP enrichment without exogenous expression of tagged proteins or specific antibodies. Because PPPs are among the most conserved proteins across evolution, PIB-MS can be employed in any cell line, tissue or organism.
Identifiants
pubmed: 34518704
doi: 10.1038/s41596-021-00604-3
pii: 10.1038/s41596-021-00604-3
pmc: PMC8822503
mid: NIHMS1774052
doi:
Substances chimiques
Phosphoprotein Phosphatases
EC 3.1.3.16
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Review
Langues
eng
Sous-ensembles de citation
IM
Pagination
4919-4943Subventions
Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : NCI NIH HHS
ID : R33 CA225458
Pays : United States
Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer Nature Limited.
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