Proteomics pipeline for phosphoenrichment and its application on a human melanoma cell model.
Talanta
; 220: 121381, 2020 Dec 01.
Article
em En
| MEDLINE
| ID: mdl-32928406
ABSTRACT
Cell signalling is tightly regulated by post-translational modification of proteins. Among them, phosphorylation is one of the most interesting and important. Identifying phosphorylation sites on proteins is challenging and requires strategies for pre-separation and enrichment of the phosphorylated species. We applied four different methods for phospho-enrichment involving TiO2 and IMAC matrix to human melanoma cell lysates of starved A375 induced for 1 h with 1% FBS. Comparison of protocol efficiency was evaluated through peptide concentration, sulphur and phosphorus content and peptide analysis by LC-MS in the collected fractions. Our results underlined that each single method is not sufficient for a comprehensive phosphoproteome analysis. In fact, each methodology permits to identify only a fraction of the phosphoproteome contained in a whole cell lysate. The selection of the most efficient protocols and a combination of two phospho-enrichment methods allowed the assessment of this workflow able to pinpoint the main actors in the phospho-proteome cascade of A375 human melanoma cells treated with Vemurafenib.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Proteômica
/
Melanoma
Limite:
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article