Candida albicans induces pro-inflammatory and anti-apoptotic signals in macrophages as revealed by quantitative proteomics and phosphoproteomics.

ABSTRACT: Proteomic analysis: A stable isotope labeling approach (SILAC) was used to study changes in protein expression from murine macrophages in response to 3h of interaction with C. albicans (1:1 ratio) . Proteins were identified by Gel-LC-MS (1D SDS-PAGE, trypsin digestion and analysis by EASY-nLC LTQ Orbitrap XL Mass Spectrometer). Protein quantitation and identification was carried out with Proteome Discoverer v1.2 software. The relative abundance of a protein was measured by averaging the intensities of all peptides identified for the protein at each sample. Three independent biological replicas and two technical replicas of each were performed. Phosphoproteomic analysis: Two different and complementary phosphopeptide enrichment approaches were performed. Equal amounts of SILAC samples from control macrophages (grown in “light medium” 12C6-arginine and 12C6-lysine) and macrophages after 3h of interaction with C. albicans (grown in “heavy medium” 13C6-arginine and 13C6-lysine) were mixed and digested prior to phosphopeptide enrichment by ion metal affinity chromatography (IMAC) combined with TiO2 enrichment (SIMAC) or by Calcium Phosphate Precipitation (CPP) combined with TiO2. Three biological and 2 technical replicas of each condition were treated. All the SIMAC and CPP samples were analyzed by EASY-nLC LTQ Orbitrap XL Mass Spectrometer. Raw data files were processed with the Proteome Discoverer v1.2 software for peptide and protein identification and the quantification of phosphorylation changes. Finally, phosphorylation site ratios measured in both technical replica experiments were averaged prior to further data analysis.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Jose Antonio Reales-Calderon  

LAB HEAD: Concha Gil

PROVIDER: PXD000406 | Pride | 2020-01-24

REPOSITORIES: Pride