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

Abstract

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 to its solvent accessibility and to the resilience of the residual protein structure in solution. Temperature-dependent digestion experiments allowed estimation of the type of secondary structures around the cleavage site.

Investigation of Structure-Stabilizing Elements in Proteins by Ion Mobility Mass Spectrometry and Collision-Induced Unfolding

“…The online enzymatic reactor used in this study was previously detailed elsewhere. 16 In summary, the designed enzymatic digestion reactor comprised two pumps: a thermostatic syringe containing the targeted protein and the enzyme (flux of 4 μL min −1 ) and a second syringe containing 10% acetonitrile + formic acid 0.2% (v/v) (flux of 4 μL min −1 ) used to quench the enzymatic digestion reaction in the mixing chamber prior to the MS analysis (see Figure S2). The enzyme was introduced to the sample just before injection to monitor the enzymatic digestion of the model proteins over time.…”

“…In our previous study, we demonstrated how the secondary structure of proteins and peptides influences the initial apparition rate 16 and time of appearance of peptides generated by a protease. The initial apparition rate of released peptides was found to be directly dependent on the protein's state (folded or denatured) and the cleavage site's location on structural domains (α-helix/β-sheet) or flexible domains.…”

“…The combination of digestion and CIU monitoring of partially digested protein structures might provide a new IMS-based approach to evaluate the structuring effect of specific domains in proteins. The reported method has been implemented on the direct coupling of an enzymatic reactor previously published by our team 16 to a traveling wave ion mobility instrument, a Synapt G2-Si HDMS (Waters Corp., Manchester, UK). As a proof of concept, the proposed approach has first been applied to a model peptide containing two disulfide bridges.…”