Covalent labeling-mass spectrometry with non-specific reagents for studying protein structure and interactions

Abstract

Using mass spectrometry (MS) to obtain information about a higher order structure of protein requires that a protein's structural properties are encoded into the mass of that protein. Covalent labeling (CL) with reagents that can irreversibly modify solvent accessible amino acid side chains is an effective way to encode structural information into the mass of a protein, as this information can be read-out in a straightforward manner using standard MS-based proteomics techniques. The differential reactivity of proteins under two or more conditions can be used to distinguish protein topologies, conformations, and/or binding sites. CL-MS methods have been effectively used for the structural analysis of proteins and protein complexes, particularly for systems that are difficult to study by other more traditional biochemical techniques. This review provides an overview of the non-specific CL approaches that have been combined with MS with a particular emphasis on the reagents that are commonly used, including hydroxyl radicals, carbenes, and diethylpyrocarbonate. We describe the reagent and protein factors that affect the reactivity of amino acid side chains. We also include details about experimental design and workflow, data analysis, recent applications, and some future prospects of CL-MS methods.

PyXlinkViewer: a flexible tool for visualisation of protein chemical crosslinking data within the PyMOL molecular graphics system

“…FPOP, it remains analytically challenging to determine at the residue level the site of the modification. Nevertheless, data from covalent labelling has shown promise as an input for structural modelling (36)(37)(38). To demonstrate the data input requirements for covalent labelling/ dead-end XL data we have also included example data from FPOP analysis of the protein β 2 -microglobulin (17) (see example data).…”

DNA binding reorganizes the intrinsically disordered C-terminal region of PSC inDrosophilaPRC1

“…These could include protein-RNA interactions, which can also be mediated by lysine-rich IDRs [42]. In principle, other amino acids involved in nucleic acid interactions could be analyze using similar footprinting approaches [43].…”

Evolution of Structural Biology through the Lens of Mass Spectrometry

“…CL-MS techniques using hydroxyl radicals, DEPC, and carbenes rely on the formation of new covalent bonds with solvent accessible amino acid side chains providing information on the proteins surface structure. 96…”