The cornerstone of mammalian intrinsic and innate immune defense against pathogens is the production of interferons (IFN), cytokines that stimulate anti-pathogen signaling pathways through the simultaneous expression of hundreds of interferon-stimulated genes (ISG). In this work, we sought to compare the effects of IFN classes on the cellular proteome. Upon classifying sets of both shared and distinct ISG proteins responsive to each IFN class, we determined the relative protein alterations using parallel reaction monitoring (PRM) by developing a proteotypic peptide library for ISG markers induced by each IFN class. Additional comparison to stimulation with pro-inflammatory LPS helped to contextualize the observed IFN signatures.

Cells were lysed in 5% SDS and 50 mM triethylammonium bicarbonate (TEAB), pH 8.5. and trypsinized using S-Trap micro spin columns (Protifi). Samples were analyzed by LC-MS/MS using a Dionex Ultimate 3000 nanoRSLC coupled online to an EASYSpray ion source and a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer. The panel assessed 177 peptides split into 4 separate scheduled PRM methods with six minute retention time windows. RAW PRM files were imported into Skyline and also searched by Proteome Discoverer and search results were imported into Skyline as a spectral library.

PMA-differentiated THP-1 cells were treated with either Type I IFN-beta, Type II IFN-gamma, or lipopolysaccharide for 24 hours. Cells were lysed in 5% SDS and 50 mM triethylammonium bicarbonate (TEAB), pH 8.5 and processed for PRM analysis of IFN-responsive proteins.