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

Abstract

A proteolytic reactor, recently designed by our group for structural investigation of proteins 1 was coupled to ion mobility mass spectrometry to follow the evolution of collision cross section (CCS) values of the residual parts of proteins subjected to mono enzymatic digestion (trypsin digest), Upon the progressive loss of various peptides during digestion, the CCS of the remaining sequence of the protein can either comply or diverge from the classical 2/3 power of the CCS-mass relationship (i.e., for spherical structures). Indeed, proteins are generally considered to adopt a globular shape in the gas phase, which correlates CCS to mass via the equation 2 CCS = A x m 2/3 . Upon the loss of stabilizing elements during digestion, the residual structure can be disrupted and the corresponding CCS value stop complying to the aforementioned trend. In complement to the determination of their CCS values, the residual structures have also been characterized using collision induced unfolding (CIU) to probe their respective resilience towards collisional activation with a neutral gas. The characterized resilience can then be linked to the presence of structure stabilizing element(s) within the proteins and related residual structures. In this study, β-lactoglobulin (2 disulfide bridges), cytochrome c (heme) and calmodulin (Ca 2+ coordination cation) were used to probe the effect of various commonly found structuring elements in proteins on the CCS values. In addition, CIU was performed on protein related residual structures to assess their resilience in the gas phase upon the loss of these various structuring elements. In the gas phase two factor are responsible of the protein unfolding: the charge and the energy barrier. By applying CIU on the structure with or without structure stabilizing elements, it is expected to have to supply more energy on the structure with stabilizing elements. The energy barrier increased in the presence of structure stabilizing elements. TW CCS N2→He of the studied species were plotted as a function of their masses and compared to two trend curves describing the CCS/mass relation: (1) a trend curve established by Ruotolo et al. 2 CCS = 2.45 x m 2/3 and (2) a similar trend curve established by our group using the trypsin digest of cytochrome c and b-lactoglobulin sprayed in non-denaturing conditions CCS = 2.39 x m 2/3 . It is generally admitted that TW CCS N2→He located below or on the trend curve reflects the presence of more compact structures while those located above the trend curve are related to more extended species.