Label-free proteomics to study the effect of IL-9 on MDA-MB-231 and HeLa cells

ABSTRACT: To study the proteomic profile of HeLa and MDA-MB-231 upon IL-9 stimulation, we treated both the cell-lines with the cytokine for 24 hours. Quantitative label-free proteomic analysis was then performed and abundance ratios were obtained for proteins from cells in IL-9 treated v. untreated conditions. Method: TRIzol (Thermo Fisher Scientific, USA, Cat. No. 15596026) reagent-based method was used for protein extraction from MDA-MB-231 and HeLa. Cells were washed with PBS and lysed in TRIzol followed by protein precipitation using manufacturers protocol. The extracted protein was re-solubilized in Urea (8 M) Thiourea (2 M) Tris-Cl Buffer. Protein was treated with 15 mM Dithiothreitol (DTT) for 1 hour at 50 deg C followed by 30 mM Iodoacetamide (IAA) treatment and digestion with trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, USA, Cat. No. V5280). Digested protein was desalted using C18 Stage tips. 500 ng peptides were injected using EasyNLC 1200 (Thermo Fisher Scientific, USA) instrument into a Q-Exactive Plus Orbitrap Mass Spectrometer with a 2-hour gradient in Data Dependent Acquisition (DDA) mode (Resolution: MS1=70000, MS2=17500). Mass Spectra were then analyzed using Proteome Discoverer 2.2 software (ThermoFisher Scientific, USA, Cat. No. XCALI-97808). Mascot and SequestHT were employed as search engines for discovery proteomics. For label-free quantification, standard workflows by the manufacturer were employed with the following specific parameters: the samples were normalized with respect to the peak-area based total peptide amount, the permitted retention time shift (RT) was set to 2 min, precursor mass tolerance to 2 ppm, fragment mass tolerance to 0.02 Da and the false discovery rate (FDR) to 0.01 (strict). Only unique peptides were included for peak-area based quantification of proteins. For statistical analysis of identified proteins, hypothesis testing was performed using background-based ANOVA. Proteins identified were filtered for high FDR confidence and only proteins with 2 or more unique peptides were considered for relative quantification and abundance ratios were calculated. Differentially expressed proteins (with at least 1.5 fold upregulation or downregulation) thus obtained were used for pathway enrichment and functional annotation analysis using DAVID bioinformatics tool, PANTHER and STRING database. Heatmaps were rendered using ClustVis Heatmap online server.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Rahul Purwar  

PROVIDER: MSV000086990 | MassIVE | Wed Mar 03 09:04:00 GMT 2021

REPOSITORIES: MassIVE