應用文集

Protein activity is mainly modulated by dynamic reversible post-translational modifications (PTMs) such as site-specific phosphorylation, which regulates all cellular processes. Despite continuous improvements, global analysis of protein phosphorylation is still challenging due to its sub-stoichiometric nature and low abundance. Off-line high pH reversed-phase peptide fractionation has shown great promise to overcome this challenge and provides a basic framework for performing in-depth phosphoproteomics studies. However, deep phosphoproteome analyses typically require mg of starting material per sample condition and several days of instrument time. This is not feasible for large-scale clinical studies with many experimental conditions, where deep phosphoproteomes from low amounts of starting material with rapid single-shot analyses are preferred. Here we describe, in detail, how to setup an efficient and reproducible workflow with automated PAC digestion and Ti-IMAC phospho-enrichment on a Kingfisher Flex robot and utilize the short and sensitive separation methods on the Evosep One in combination with fast scanning MS/MS methods on an Orbitrap Exploris 480 MS.