Cation-induced stabilization of protein complexes in the gas phase: mechanistic insights from hemoglobin dissociation studies.

Collision-induced dissociation (CID) of electrosprayed protein complexes usually involves asymmetric charge partitioning, where a single unfolded chain gets ejected that carries a disproportionately large fraction of charge. Using hemoglobin (Hb) tetramers as model system, we confirm earlier reports that bound metal ions can stabilize protein complexes under CID conditions. We examine the mechanism underlying this effect. Nonvolatile salts cause extensive adduct formation. Significant stabilization was observed for Mg(2+) and Ca(2+), whereas K(+), Rb(+), and Cs(+) had no effect. Precursor ion selection was used to examine Hb subpopulations with well-defined metal binding levels. K(+), Rb(+), and Cs(+)-adducted tetramers eject monomers that carry roughly one-quarter of the metal ions that were bound to the precursor. This demonstrates that charge migration during CID is exclusively due to proton transfer, not metal ion transfer. Also, replacement of highly mobile charge carriers (protons) with less mobile species (metal ions) does not exert a stabilizing influence under the conditions used here. Interestingly, Hb carrying stabilizing ions (Mg(2+) and Ca(2+)) generates monomeric CID products that are metal depleted. This effect is attributed to a combination of two factors: (1) Me(2+) binding stabilizes Hb via formation of chelation bridges (e.g., R-COO(-) Me(2+) (-)OOC-R); the more Me(2+) a subunit contains the more stable it is. (2) More than ~90% of the tetramers contain at least one subunit with a below-average number of Me(2+). The prevalence of monomeric CID products with depleted Me(2+) levels is caused by the tendency of these low metal-containing subunits to undergo preferential unfolding/ejection.