Ion mobility-resolved phosphoproteomics with dia-PASEF and short gradients.
Proteomics
; 23(7-8): e2200032, 2023 04.
Article
em En
| MEDLINE
| ID: mdl-36300730
ABSTRACT
Mass spectrometry-based phosphoproteomics has identified >150,000 post-translational phosphorylation sites in the human proteome. To disentangle their functional relevance, complex experimental designs that require increased throughput are now coming into focus. Here, we apply dia-PASEF on a trapped ion mobility (TIMS) mass spectrometer to analyze the phosphoproteome of a human cancer cell line in short liquid chromatography gradients. At low sample amounts equivalent to â¼20 ug protein digest per analysis, we quantified over 13,000 phosphopeptides including â¼8700 class I phosphosites in 1 h without a spectral library. Decreasing the gradient time to 15 min yielded virtually identical coverage of the phosphoproteome, and with 7 min gradients we still quantified about 80% of the class I sites with a median coefficient of variation <10% in quadruplicates. We attribute this in part to the increased peak capacity, which effectively compensates for the higher peptide density per time unit in shorter gradients. Our data show a five-fold reduction in the number of co-isolated peptides with TIMS. In the most extreme case, these were positional isomers of nearby phosphosites that remained unresolved with fast liquid chromatography. In summary, our study demonstrates how key features of dia-PASEF translate to phosphoproteomics.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Fosfopeptídeos
/
Proteoma
Limite:
Humans
Idioma:
En
Revista:
Proteomics
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Alemanha