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Abstract :
[en] Since the apparition of conservative soft-ionization sources in the late 80s, i.e. electrospray (ESI) and matrix-assisted laser desorption-ionization, mass spectrometry (MS) has been extensively used to identify, quantify and characterize wide types of biomolecules. Especially, ESI sources make feasible the transfer of intact biomolecules and macromolecular complexes in the mass spectrometer, opening the so-called field of “native” MS. However, major questioning and inherent skepticism yet surround the native MS field concerning the ability to preserve non-bonded biological interactions during and after the molecular transfer from the physiological aqueous media to the gas-phase. Indeed, the introduction of biological structures into the gas-phase may eventually result in a reshape of their tridimensional fold, therefore leading to a misinterpretation of further derived structural data.
In an effort to circumvent gas-phase unfolding scenarios, we here suggest a structural strategy relying on intra-molecular covalent cross-linkers acting as molecular scaffolds. We focused on small 10 kDa to 20 kDa proteins, such as cytochrome c, myoglobin and β-lactoglobulin, which are yet often used as model systems to gauge wide range of structural, kinetic and thermodynamics methodologies. We studied the products of “type 0” and “type 1” cross-linking reactions achieved using bissulfosuccinimidyl suberate (BS3) as a linker reagent and compared them with their non-linked counter-parts regarding their respective structural properties. The model systems were structurally investigated both in solution, through circular dichroism and thermal denaturation measurements, and in the gas-phase following ESI injection, through IM-MS and collision induced unfolding (CIU) measurements. The gas-phase data were acquired from different solvent conditions and compared with “native-compliant” benchmarks furnished by NMR spectroscopy.
In general, the content in secondary structures within the proteins was found barely unaffected by the presence of the linkers when operating in physiological solution, therefore witnessing a preservation of the native fold upon linking reaction. However, after ionization and transfer into the gas-phase, we monitored significant reshapes in the conformational landscapes materialized through a shift of the collision cross-sections distribution towards lower values when linkers are present. The magnitude of the compaction effect is directly dependent on the protein charge and linker content. Altogether, the comprehension of the fate of electro-sprayed cross-linked conformers in the gas-phase and their respective correlation with the physiological fold achieved in solution constitute major steps in the validation of this technique as part of native MS workflows.