AP-MS analysis of IFIT1 and 5 interactions in interferon-stimulated human cells
This project sought to identify interactions of InterFeron Induced Tetratricopeptide repeat (IFIT) proteins 1 ad 5 in human cells. SILAC-labelled human HEK293T cells were transfected with FLAG-tagged human IFIT1 and IFIT5 pulldowns. After transfection, the cells were treated with human interferon 2-alpha and FLAG-immunoprecipitations performed to identify IFIT-binding proteins. Cells were then subject to LS/MS-MS analysis on a Orbitrap Fusion and data analysed in Maxquant v184.108.40.206
Sample Processing Protocol
HEK293T cells were cultured in Arg/Lys-free DMEM, supplemented with light (R0K0, medium (R6K4) or heavy (R10K8) amino acids and grown for >5 cell divisions to ensure complete labelling. Cells were transfected with the relevent plasmid using lipofectamine 2000. The experiment was performed in triplicate. Light cells were transfected with the control empty pCDNA3.1 vector, 2/3 of the medium cells were transfected with pCDNA3.1 IFIT1-FLAG, and 1/3 with pCDNA3.1 IFIT5-FLAG. 2/3 heavy cells were transfected with pCDNA3.1 IFIT5-FLAG and 1/3 with pCDNA3.1 IFIT1-FLAG. 24h post-transfection the media was replaced with fresh media containing 1000U/mL human interferon 2 alpha. Cells were lysed and samples immunoprecipitated with M2 anti-FLAG beads (Sigma) according to the manufactorers recommended protocol and submitted to the University of Bristol Proteomics facility for analysis. The samples were subjected to SDS-PAGE elctrophoesis on a precast gel, and extracted as a single band for in-gel trypsinisation. The resulting peptides were fractionated using an Ultimate 3000 nanoHPLC system in line with an LTQ Orbitrap Fusion Tribrid mass spectrometer.
Data Processing Protocol
The raw data files were processed and quantified using Maxquant v220.127.116.11 and searched against the Uniprot Human database (70,550 entries, dated 19th September 2016) using the built-in Andromeda search engine. Peptide precursor mass tolerance was set a 4.5ppm, and MS/MS tolerance was set at 0.5Da. Search criteria included carbaminomethylation of cysteine as a fixed modification. Oxidation of methionine and N-terminal acetylation were selected as variable modifications.
Fleith RC, Mears HV, Leong XY, Sanford TJ, Emmott E, Graham SC, Mansur DS, Sweeney TR. IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA. Nucleic Acids Res. 2018 PubMed: 29554348