Sample preparation for TMT-based total and phospho-proteomic analysis of cells and tissues

Mass spectrometry-based proteomics and phosphoproteomics are highly sensitive and un-biased techniques to study the proteome and phosphoproteome at a global scale. Sample preparation is a key element for the generation of high quality, reproducible data. Here we provide a step-by-step protocol for processing material derived from cells or tissue samples. We recommend employing S-Trap assisted tryptic digestion followed by a TiO2-based phosphopeptide enrichment to achieve the highest possible reproducibility across experimental replicates. We also provide 10 or 16 plex Tandem Mass Tags (TMT) multiplexing strategy in combination with High-pH reversed-phase fractionation to achieve high coverage for phosphoproteomic analysis. The nano-liquid chromatography and High-resolution mass spectrometry instrument settings for both MS2 and Synchronous precursor selection MS3 data acquisition on Orbitrap Lumos Tribrid mass spectrometer are also described. Using these protocols, we routinely identify and quantify >35,000 phosphosites and ~10,000 protein groups.