Global, non-targeted proteomic strategies are based on the systematic analysis of complex protein mixtures. As opposed to targeted MRM approaches (which rely on the monitoring of a specific panel of candidate proteins), such hypothesis-free, global methods allow a wide characterization of protein samples and have a large application scope in discovery studies. They are indeed widely used for the analysis of protein complexes and the identification of new protein interactions, the characterization of post-translational modifications, or the large-scale analysis of protein expression profiles. Approaches based on high-throughput peptide sequencing by nanoLC-MS/MS analysis benefited in recent years from major improvements in the sequencing-speed, resolution, and dynamic range of modern mass spectrometers, and proved to be powerful for the in-depth analysis of whole proteomes.
The WP4 of ProFi is dedicated to the optimization of all the analytical steps involved in such global proteomics workflows. At present, two major challenges in these strategies remain to be addressed: 1/ the improvement of the analytical coverage for highly complex samples, in order to increase the depth of the proteomic analysis and reach the identification of minor components, and 2/ the performances of quantitative analysis in such large-scale studies. While the optimization of proteomic depth is more directly linked to upstream analytical steps (sample treatment, chromatographic setup, mass spectrometry data acquisition), the accuracy of quantitative analysis using recent â€œlabel-freeâ€ methods is largely dependent on the bioinformatics processing of the MS data. Using dedicated protein standards, we are working on the optimization of both analytical and bioinformatic processes, in order to set-up efficient methods for large-scale quantitative proteomic analysis.