Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition without the need for spectral libraries.
Nat Commun
; 11(1): 787, 2020 02 07.
Article
de En
| MEDLINE
| ID: mdl-32034161
ABSTRACT
Quantitative phosphoproteomics has transformed investigations of cell signaling, but it remains challenging to scale the technology for high-throughput analyses. Here we report a rapid and reproducible approach to analyze hundreds of phosphoproteomes using data-independent acquisition (DIA) with an accurate site localization score incorporated into Spectronaut. DIA-based phosphoproteomics achieves an order of magnitude broader dynamic range, higher reproducibility of identification, and improved sensitivity and accuracy of quantification compared to state-of-the-art data-dependent acquisition (DDA)-based phosphoproteomics. Notably, direct DIA without the need of spectral libraries performs close to analyses using project-specific libraries, quantifying > 20,000 phosphopeptides in 15 min single-shot LC-MS analysis per condition. Adaptation of a 3D multiple regression model-based algorithm enables global determination of phosphorylation site stoichiometry in DIA. Scalability of the DIA approach is demonstrated by systematically analyzing the effects of thirty kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that specific protein kinases mediate EGF-dependent phospho-regulation.
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Phosphopeptides
/
Protein kinases
/
Algorithmes
/
Biologie informatique
/
Protéomique
/
Spectrométrie de masse en tandem
Type d'étude:
Prognostic_studies
Limites:
Humans
Langue:
En
Journal:
Nat Commun
Sujet du journal:
BIOLOGIA
/
CIENCIA
Année:
2020
Type de document:
Article
Pays d'affiliation:
Danemark