Charge-State-Dependent Variation of Signal Intensity Ratio between Unbound Protein and Protein–Ligand Complex in Electrospray Ionization Mass Spectrometry: The Role of Solvent-Accessible Surface Area

Native electrospray
ionization mass spectrometry (ESI-MS) is nowadays
widely used for the direct and sensitive determination of protein
complex stoichiometry and binding affinity constants (Ka). A common yet poorly understood phenomenon in native
ESI-MS is the difference between the charge-state distributions (CSDs)
of the bound protein–ligand complex (PL) and unbound protein
(P) signals. This phenomenon is typically attributed to experimental
artifacts such as nonspecific binding or in-source dissociation and
is considered highly undesirable, because the determined Ka values display strong variation with charge state. This
situation raises serious concerns regarding the reliability of ESI-MS
for the analysis of protein complexes. Here we demonstrate that, contrary
to the common belief, the CSD difference between P and PL ions can
occur without any loss of complex integrity, simply due to a change
in the solvent-accessible surface area (ΔSASA) of the protein
upon ligand binding in solution. The experimental CSD shifts for PL
and P ions in ESI-MS are explained in relation to the magnitude of ΔSASA
for diverse protein–ligand systems using a simple model based
on the charged residue mechanism. Our analysis shows that the revealed
ΔSASA factor should be considered rather general and be given
attention for the correct spectral interpretation of protein complexes.