Oxidized and Reduced Dimeric Protein Complexes Illustrate Contrasting CID and SID Charge Partitioning

Charge partitioningduring the dissociation of protein complexesin the gas phase is influenced by many factors, such as interfacialinteractions, protein flexibility, protein conformation, and dissociationmethods. In the present work, two cysteine-containing homodimer proteins,& beta;-lactoglobulin and & alpha;-lactalbumin, with the disulfidebonds intact and reduced, were used to gain insight into the chargepartitioning behaviors of collision-induced dissociation (CID) andsurface-induced dissociation (SID) processes. For these proteins,we find that restructuring dominates with CID and dissociation withsymmetric charge partitioning dominates with SID, regardless of whetherintramolecular disulfide bonds are oxidized or reduced. CID of thecharge-reduced dimeric protein complex leads to a precursor with aslightly smaller collision cross section (CCS), greater stability,and more symmetrically distributed charges than the significantlyexpanded form produced by CID of the higher charged dimer. Collision-inducedunfolding plots demonstrate that the unfolding-restructuringof the protein complexes initiates the charge migration of highercharge-state precursors. Overall, gas collisions reveal the charge-dependentrestructuring/unfolding properties of the protein precursor, whilesurface collisions lead predominantly to more charge-symmetric monomerseparation. CID's multiple low-energy collisions sequentiallyreorganize intra- and intermolecular bonds, while SID's large-stepenergy jump cleaves intermolecular interfacial bonds in preferenceto reorganizing intramolecular bonds. The activated population ofprecursors that have taken on energy without dissociating (populatedin CID over a wide range of collision energies, populated in SID foronly a narrow distribution of collision energies near the onset ofdissociation) is expected to be restructured, regardless of the activationmethod.

Automated summary

The charge partitioning during the dissociation of protein complexes in the gas phase is influenced by various factors, including interfacial interactions, protein flexibility, protein conformation, and dissociation methods. This study used two cysteine-containing homodimer proteins to investigate the charge partitioning behaviors of collision-induced dissociation (CID) and surface-induced dissociation (SID) processes. Results showed that restructuring dominates with CID, while dissociation with symmetric charge partitioning dominates with SID, regardless of the oxidation state of the intramolecular disulfide bonds. Gas collisions reveal the charge-dependent restructuring/unfolding properties of the protein precursor, while surface collisions predominantly lead to more charge-symmetric monomer separation.