Reference: Valk E, et al. (2020) Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Quantitative Mass-Spectrometry. Methods Mol Biol 2141:819-833

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Abstract


Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) within proteins have attracted considerable attention in recent years. Several important biological signaling mechanisms including protein-protein interactions and post-translational modifications can be easily mediated by IDPs and IDRs due to their flexible structure. These regions can encode linear sequences that are indispensable in cell-signaling networks and circuits. For example, the linear multisite phosphorylation networks encoded in disordered protein sequences play a key role in cell-cycle regulation where the phosphorylation of proteins controls the orchestration of all major mechanisms. While elucidating a systems-level understanding of this process and other multisite phosphorylation processes, we extensively used mass-spectrometry and found it to be an ideal tool to identify, characterize, and quantify phosphorylation dynamics within IDPs. Here, we describe a quantitative proteomics method, together with a detailed protocol to analyze dynamic multisite phosphorylation processes within IDPs using an in vitro protein phosphorylation assay with "light" gamma-16O ATP and "heavy" gamma-18O ATP, combined with liquid chromatography mass spectrometry.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't
Authors
Valk E, Maljavin A, Loog M
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