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Label-Free Higher Order Structure and Dynamic Investigation Method of Proteins in Solution Using an Enzymatic Reactor Coupled to Electrospray High-Resolution Mass Spectrometry Detection
For decades, structural analysis of proteins have received considerable attention, from their sequencing to the determination of their 3D structures either in the free state (e.g., no host–guest system, apoproteins) or (non)covalently bound complexes. The elucidation of the 3D structures and the ma...
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| Published in: | Journal of the American Society for Mass Spectrometry 2022-02, Vol.33 (2), p.284-295 |
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| Main Authors: | , , , , , , |
| Format: | Article |
| Language: | English |
| Subjects: | |
| Citations: | Items that this one cites Items that cite this one |
| Online Access: | Get full text |
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| Summary: | For decades, structural analysis of proteins have received considerable attention, from their sequencing to the determination of their 3D structures either in the free state (e.g., no host–guest system, apoproteins) or (non)covalently bound complexes. The elucidation of the 3D structures and the mapping of intra- and intermolecular interactions are valuable sources of information to understand the physicochemical properties of such systems. X-ray crystallography and nuclear magnetic resonance are methods of choice for obtaining structures at the atomic level. Nonetheless, they still present drawbacks which limit their use to highly purified systems in a relatively high amount. On the contrary, mass spectrometry (MS) has become a powerful tool thanks to its selectivity, sensitivity, and the development of structural methods both at the global shape and the residue level. The combination of several MS-based methods is mandatory to fully assign a putative structure in combination with computational chemistry and bioinformatics. In that context, we propose a strategy which complements the existing methods of structural studies (e.g., circular dichroism, hydrogen/deuterium exchange and cross-links experiments, nuclear magnetic resonance). The workflow is based on the collection of structural information on proteins from the apparition rates and the time of appearance of released peptides generated by a protease in controlled experimental conditions with online detection by electrospray high-resolution mass spectrometry. Nondenaturing, partially or fully denatured proteins were digested by the enzymatic reactor, i.e., β-lactoglobulin, cytochrome c, and β-casein. The collected data are interpreted with regard to the kinetic schemes with time-dependent rates of the enzymatic digestion established beforehand, considering kinetics parameters in the Michaelis–Menten formalism including k cat (the turnover number), k 1 (formation of the enzyme–substrate complex), k –1 (dissociation of the enzyme–substrate complex), k off (local refolding of the protein around the cleavage site), and k on (local unfolding of the protein around the cleavage site). Solvent-accessible surface analysis through digestion kinetics was also investigated. The initial apparition rates of released peptides varied according to the protein state (folded vs denatured) and informs the k off/k on ratio around the cleavage site. On the other hand, the time of appearance of a given peptide is related |
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| ISSN: | 1044-0305 1879-1123 |
| DOI: | 10.1021/jasms.1c00274 |