Abstract
Structural heterogeneity is a significant challenge complicating (and in some cases making impossible) electrospray ionization mass spectrometry (ESI MS) analysis of noncovalent complexes comprising structurally heterogeneous biopolymers. The broad mass distribution exhibited by such species inevitably gives rise to overlapping ionic signals representing different charge states, resulting in a continuum spectrum with no discernible features that can be used to assign ionic charges and calculate their masses. This problem can be circumvented by using limited charge reduction, which utilizes gas-phase chemistry to induce charge-transfer reactions within ionic populations selected within narrow m/z windows, thereby producing well-defined and readily interpretable charge ladders. However, the ionic signal in native MS typically populates high m/z regions of mass spectra, which frequently extend beyond the precursor ion isolation limits of most commercial mass spectrometers. While the ionic signal of single-chain proteins can be shifted to lower m/z regions simply by switching to a denaturing solvent, this approach cannot be applied to noncovalent assemblies due to their inherent instability under denaturing conditions. An alternative approach explored in this work relies on adding supercharging reagents to protein solutions as a means of increasing the extent of multiple charging of noncovalent complexes in ESI MS without compromising their integrity. This shifts the ionic signal down the m/z scale to the region where ion selection and isolation can be readily accomplished with a front-end quadrupole, followed by limited charge reduction of the isolated ionic population. The feasibility of the new approach is demonstrated using noncovalent complexes formed by hemoglobin with structurally heterogeneous haptoglobin.