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Abstract
Use of a heterobifunctional photoactivatable cross-linker, sulfo-SDA (diazirine), has yielded high-density data that facilitated structure modeling of individual proteins. We expand the photoactivatable chemistry toolbox here with a second reagent, sulfo-SBP (benzophenone). This further increases the density of photo-cross-linking to a factor of 20× over conventional cross-linking. Importantly, the two different photoactivatable groups display orthogonal directionality, enabling access to different protein regions, unreachable with a single cross-linker.
Highlights
Cross-linking/mass spectrometry (CLMS) is a widespread method for investigating protein structure and interactions
These linkages are detected by mass spectrometry and function as distance constraints when modeling the structure
Data-density of identified distance constraints is curtailed by the restricted specificity of homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers predominantly used in CLMS analysis, limiting the level of protein structure detail obtainable
Summary
Cross-linking/mass spectrometry (CLMS) is a widespread method for investigating protein structure and interactions. Selective cross-linking chemistry is one factor that limits the current resolution of this approach.[1] In CLMS, new covalent bonds are introduced between atoms near in space but not necessarily close in the sequence of the protein. These linkages are detected by mass spectrometry and function as distance constraints when modeling the structure. Benzophenones have previously been used to capture protein interactions in the context of amino acid analogue incorporation, but this has typically involved targeting single incorporation sites, limiting the extent of identified links.[16−25] A previous study aimed at comparing exogenous bifunctional cross-linkers containing different photoactivatable groups, including diazirine and benzophenone. We aimed to establish whether the use of a benzophenone based cross-linker could result in a high number of crosslinks and whether cross-linking site selection would be influenced by the different chemical nature of the two photoactivatable groups
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