Proteomics pipeline for phosphoenrichment and its application on a human melanoma cell model

Talanta. 2020 Dec 1;220:121381. doi: 10.1016/j.talanta.2020.121381. Epub 2020 Jul 17.

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.

Keywords: BRAF inhibitor; ICP-MS; LC-MS/MS; Phosphoproteomics.

MeSH terms

  • Chromatography, Liquid
  • Humans
  • Melanoma* / drug therapy
  • Phosphopeptides / metabolism
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Proteome / metabolism
  • Proteomics*
  • Tandem Mass Spectrometry

Substances

  • Phosphopeptides
  • Phosphoproteins
  • Proteome