On the mechanism of protein supercharging in electrospray ionisation mass spectrometry: Effects on charging of additives with short- and long-chain alkyl constituents with carbonate and sulphite terminal groups

Small organic molecules are used as solution additives in electrospray ionisation mass spectrometry (ESI-MS) to increase the charge states of protein ions and improve the performance of intact protein analysis by tandem mass spectrometry. The properties of the additives that are responsible for their charge-enhancing effects (e.g. dipole moment, gas-phase basicity, Brønsted basicity, and surface tension) have been debated in the literature. We report a series of solution additives for ESI-MS based on cyclic alkyl carbonates and sulphites that have alkyl chains that are from two to ten methylene units long. The extent of charging of [Val [5]]-angiotensin II, cytochrome c, carbonic anhydrase II, and bovine serum albumin in ESI-MS using the additives was measured. For both the alkyl carbonate and sulphite additives with up to four methylene units, ion charging increased as the side chain lengths of the additives increased. At a critical alkyl chain length of four methylene units, protein ion charge states decreased as the chain length increased. The dipole moments, gas-phase basicity values, and Brønsted basicities (i.e. the pK_a of the conjugate acids) of the additives were obtained using electronic structure calculations, and the surface tensions were measured by pendant drop tensiometry. Because the dipole moments, gas-phase basicities, and pK_a values of the additives did not depend significantly on the alkyl chain lengths of the additives and the extent of charging depended strongly on the chain lengths, these data indicate that these three additive properties do not correlate with protein charging under these conditions. For the additives with alkyl chains at or above the critical length, the surface tension of the additives decreased as the length of the side chain decreased, which correlated well with the decrease in protein charging. These data are consistent with protein charging being limited by droplet surface tension below a threshold surface tension for these additives. For additives with relatively high surface tensions, protein ion charging increased as the amphiphilicity of the additives increased (and surface tension decreased) which is consistent with protein charging being limited by the emission of charge carriers from highly charged ESI generated droplets.