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

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Abstract
A recently developed proteolytic reactor, designed for protein structural investigation, was coupled to ion mobility mass spectrometry to monitor collisional cross section (CCS) evolution of model proteins undergoing trypsin-mediated mono enzymatic digestion. As peptides are released during digestion, the CCS of the remaining protein structure may deviate from the classical 2/3 power of the CCS-mass relationship for spherical structures. The classical relationship between CCS and mass (CCS = A x M-2/3) for spherical structures, assuming a globular shape in the gas phase, may deviate as stabilizing elements are lost during digestion. In addition, collision-induced unfolding (CIU) experiments on partially digested proteins provided insights into the CCS resilience in the gas phase to ion activation, potentially due to the presence of stabilizing elements. The study initially investigated a model peptide ModBea (3 kDa), assessing the impact of disulfide bridges on CCS resilience in both reduced and oxidized forms. Subsequently, beta-lactoglobulin (2 disulfide bridges), calmodulin (Ca2+ coordination cation), and cytochrome c (heme) were selected to investigate the influence of common structuring elements on CCS resilience. CIU experiments probed the unfolding process, evaluating the effect of losing specific peptides on the energy landscapes of partially digested proteins. Comparisons of the (CCSN2 -> He)-C-TW to trend curves describing the CCS/mass relationship revealed that proteins with structure-stabilizing elements consistently exhibit (CCSN2 -> He)-C-TW and greater resilience toward CIU compared to proteins lacking these elements. The integration of online digestion, ion mobility, and CIU provides a valuable tool for identifying structuring elements in biopolymers in the gas phase.
Funding Information
  • European Regional Development Fund
  • Fonds De La Recherche Scientifique - FNRS
  • GlycoCell project

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