Abstract

Previously, we have reported the stabilization effect of Hofmeister salts for multiprotein complexes (MPC) in the absence of bulk solvent (J. Am. Chem. Soc.2011,133 (29), 11,358–11367; Angew. Chem. Int. Ed.2012,51 (23), 5692–5695; Angew. Chem. Int. Ed.2013, 52 (32), 8329–8332.). Our efforts sought to bridge the gap between gas-phase protein structures and those found in solution. To reveal more detailed MPC topology information, native ion mobility-mass spectrometry (IM-MS) measurements are often combined with gas-phase activation methods. Conventional activation methods, including collision induced dissociation/unfolding (CID/CIU), however, primarily report information focused on monomeric subunits within the MPC, limiting the topological information obtained. Herein, we describe a simple buffer-doping method that promotes an alternative MPC CID pathway which readily produces product ions that correspond to larger sub-complexes from within some parent assemblies. Interestingly, tetramers exhibiting a dimer of dimers quaternary structure (e.g. hemoglobin and concanavalin A) produce dimeric product ions upon collisional activation following ionization from bicarbonate buffer, in contrast to the commonly observed monomer-ejection CID pathway. In order to both further investigate and validate our native IM-MS, we performed bottom-up proteomics experiments on MPCs housed in bicarbonate buffer. Our efforts revealed evidence of bicarbonate-mediated disulfide bond formation in proximal Cystine residues. We close by discussing the applications for these observations in the context of MPC structure determination by native IM-MS.

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