Proteomics pipeline for phosphoenrichment and its application on a human melanoma cell model
- Resource Type
- Authors
- Francesco Finamore; Antonella Cecchettini; Giovanni Signore; Nadia Ucciferri; Marianna Vitiello; Laura Poliseno; Elisa Ceccherini; Silvia Rocchiccioli
- Source
- Subject
- Phosphopeptides
Proteomics
Cell signaling
Proteome
Peptide
02 engineering and technology
Computational biology
01 natural sciences
Analytical Chemistry
Tandem Mass Spectrometry
medicine
Humans
Phosphorylation
Vemurafenib
Melanoma
chemistry.chemical_classification
Phosphoproteomics ICP-MS LC-MS/MS BRAF inhibitor
Chemistry
010401 analytical chemistry
Cell model
Phosphoproteomics
021001 nanoscience & nanotechnology
Phosphoproteins
0104 chemical sciences
Human melanoma
0210 nano-technology
medicine.drug
Chromatography, Liquid
- Language
- English
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.