µPhos: a scalable and sensitive platform for high-dimensional phosphoproteomics.
Drug Response
Mass Spectrometry
Phosphoproteomics
Sample Preparation
Signaling
Journal
Molecular systems biology
ISSN: 1744-4292
Titre abrégé: Mol Syst Biol
Pays: Germany
ID NLM: 101235389
Informations de publication
Date de publication:
21 Jun 2024
21 Jun 2024
Historique:
received:
25
08
2023
accepted:
11
06
2024
revised:
06
06
2024
medline:
22
6
2024
pubmed:
22
6
2024
entrez:
21
6
2024
Statut:
aheadofprint
Résumé
Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to perturbations is crucial for further illuminating the functionality of the phosphoproteome. Here we describe µPhos ('microPhos'), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture and small tissue amounts in <8 h total processing time. By greatly minimizing transfer steps and liquid volumes, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify ~17,000 Class I phosphosites in a human cancer cell line using 20 µg starting material, and confidently localize ~6200 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable. We employ µPhos to study drug- and time-dependent response signatures in a leukemia cell line, and by quantifying 30,000 Class I phosphosites in the mouse brain we reveal distinct spatial kinase activities in subregions of the hippocampal formation.
Identifiants
pubmed: 38907068
doi: 10.1038/s44320-024-00050-9
pii: 10.1038/s44320-024-00050-9
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : Freistaat Thüringen (Free State of Thuringia)
ID : #2018 IZN 002
Organisme : Deutsche Forschungsgemeinschaft (DFG)
ID : RTG 2155
Organisme : DHAC | National Health and Medical Research Council (NHMRC)
ID : 2011083
Informations de copyright
© 2024. The Author(s).
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