Proteomics approaches for the analysis of complex proteomic mixtures and their application to the study of inflammatory and immunological responses

High-resolution mass spectrometry (MS)-based proteomics has progressed tremendously in recent years, and represents now a powerful tool to efficiently characterize highly complex protein mixtures. Classical workflows are based on the analysis of peptides derived from enzymatic digestion of proteins through liquid nanochromatography combined with online electrospray MS measurement and tandem MS (MS/MS)for high-throughput sequencing. Due to the higher sensitivity, resolution, and sequencing speed of modern instruments, the analytical coverage of protein mixtures by nanoLC-MS/MS has been largely improved, however several analytical challenges remain such as quantitative analysis, bioinformatic processing of the data, and depth of proteomic analysis when dealing with highly complex samples such as biological fluids or entire cellular proteomes. I will present an illustration on several biological projects of how proteomics can be used for characterization of protein-protein interactions and protein complexes, or quantitative analysis of global protein expression changes in large-scale studies.Affinity purification methods coupled to MS analysis were particularly used to characterize protein complexes and identify new binding partners of key signaling molecules downstream of the T-cell receptor pathway in murine T lymphocytes. We alsodevelopeda quantitative proteomic workflow for the proteomic analysis of entire cellular proteomesbased on nanoLC-MS/MS analysis on an LTQ-Orbitrap-VELOS mass spectrometer and label-free quantification using the MFPaQ software, and evaluated its performances in terms of reproducibility and number of quantified proteins, with or without protein fractionation. The method was applied for the large-scale quantitative analysis of primary human vascular endothelial cells stimulated by proinflammatory cytokines such as TNFα, INFβ and IL1β, or the more recently discovered cytokine IL-33. These studies provided an extensive characterization of the endothelial cell proteome to a depth of more than 5000 proteins and allowed to decipher the inflammatory response of these cells.