Photo-induced cross-linking (PIC) is a powerful strategy for generating information on biomolecular interactions. In PIC, the utility of traditional cross-linking methods is supplemented by the temporal control of photo-activation, enabling the study of non-covalent kinetic intermediates and heterogeneous mixtures. This tutorial review will introduce the photochemistry of activation, reactive intermediates, methods for the functionalisation of biomolecules and the installation of additional functionalities (e.g., affinity tags). In doing so, we shall illustrate the wealth of data that can be obtained using this approach, ranging from the identification of interacting partners and structural data to temporal information. Alongside a discussion of the strengths and weaknesses of the various approaches, their applicability to different types of biological system will be described.
β-Sheet peptide nanostructures (e.g., amyloid fibrils) are recognized as important entities in biological systems and as functional materials in their own right. Their unique physical properties and architectural complexity, however, present a challenge for structure determination at atomic resolution. Covalent cross-linking and mass spectrometry are appealing methods for this endeavor because, potentially, a large amount of information can be extracted from a small sample in a single experiment. Previously, we described preliminary studies on the use of a photoreactive diazirine-containing amino acid to cross-link peptide monomers in nanostructures, together with the integrated separation and analysis of the products using ion mobility spectrometry coupled to conventional mass spectrometry. Here, a pH-switchable system (Aβ(16-22), a sequence from the amyloid-β peptide) was used to examine cross-linking chemistry in morphologically distinct supramolecular structures containing, or entirely composed of, diazirine-functionalized peptides. We examine the relationship between cross-linker chemistry, covalent cross-links (identified using chemical derivatization and tandem mass spectrometry), and noncovalent structure, and report differences in the site of cross-linking that can only be explained by supramolecular templating. The results demonstrate the applicability of the approach for obtaining structural restraints in ordered supramolecular assemblies, provided that a considered evaluation of the cross-linked products is undertaken.
The emergence of novel coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 coronavirus, has necessitated the urgent development of new diagnostic and therapeutic strategies. Rapid research and development, on an international scale, has already generated assays for detecting SARS-CoV-2 RNA and host immunoglobulins. However, the complexities of COVID-19 are such that fuller definitions of patient status, trajectory, sequelae, and responses to therapy are now required. There is accumulating evidence—from studies of both COVID-19 and the related disease SARS—that protein biomarkers could help to provide this definition. Proteins associated with blood coagulation (D-dimer), cell damage (lactate dehydrogenase), and the inflammatory response (e.g., C-reactive protein) have already been identified as possible predictors of COVID-19 severity or mortality. Proteomics technologies, with their ability to detect many proteins per analysis, have begun to extend these early findings. To be effective, proteomics strategies must include not only methods for comprehensive data acquisition (e.g., using mass spectrometry) but also informatics approaches via which to derive actionable information from large data sets. Here we review applications of proteomics to COVID-19 and SARS and outline how pipelines involving technologies such as artificial intelligence could be of value for research on these diseases.
Photoinduced cross-linking (PIC) has become a powerful tool in chemical biology for the identification and mapping of stable or transient interactions between biomacromolecules and their (unknown) ligands. However, the value of PIC for in vitro and in vivo structural proteomics can be realized only if cross-linking reports accurately on biomacromolecule secondary, tertiary, and quaternary structures with residue-specific resolution. Progress in this area requires rigorous and comparative studies of PIC reagents, but despite widespread use of PIC, these have rarely been performed. The use of PIC to report reliably on noncovalent structure is therefore limited, and its potentials have yet to be fully realized. In the present study, we compared the abilities of three probes, phenyl trifluoromethyldiazirine (TFMD), benzophenone (BP), and phenylazide (PA), to record structural information within a biomolecular complex. For this purpose, we employed a self-assembled amyloid-like peptide nanostructure as a tightly and specifically packed model environment in which to photolyze the reagents. Information about PIC products was gathered using mass spectrometry and ion mobility spectrometry, and the data were interpreted using a mechanism-oriented approach. While all three PIC groups appeared to generate information within the packed peptide environment, the data highlight technical limitations of BP and PA. On the other hand, TFMD displayed accuracy and generated straightforward results. Thus TFMD, with its robust and rapid photochemistry, was shown to be an ideal probe for cross-linking of peptide nanostructures. The implications of our findings for detailed analyses of complex systems, including those that are transiently populated, are discussed.
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