Probing Metal Ion Adduction in the ESI Charged Residue Mechanism via Gas-Phase Ion/Ion Chemistry.

The final stages of the charged residue mechanism/model (CRM) for ion generation via electrospray ionization (ESI) involves the binding of excess charge onto analyte species. Ions of both polarities can bind to the analyte with an excess of ions of the same polarity as the droplet. For large biomolecule/biocomplex ions, which are commonly the species of interest in native mass spectrometry (MS), the binding of acids and salts onto the analyte can lead to extensive broadening of ion signals due to adduction. Therefore, heating step(s) to facilitate desolvation and salt adduct removal are commonplace. In this work, we describe an approach to study the final stages of CRM using gas-phase ion/ion reactions to generate analyte ion/salt clusters of well-defined composition, followed by gas-phase collision-induced dissociation (CID). While there are many variables that can be studied systematically via this approach, the work described herein is focused on salt clusters of the form [Na 10 X 11 ] - , where X = acetate (Ac - ), chloride (Cl - ), or nitrate (NO 3 - ), in reaction with a common charge state of ubiquitin as well as several model peptides. Experiments in which equimolar quantities of each salt (i.e., NaAc, NaCl, and NaNO 3 ) are subjected to ESI with ubiquitin (Ubi) and gas-phase ion/ion reaction studies involving [Na 10 X 11 ] - and [Ubi + 6H] 6+ show similar trends, in terms of the extent of sodium ion incorporation into the protein ions. Ion/ion reaction studies using model peptides show that the acetate-containing salt transfers significantly more Na + ions into the peptide ions. Exchange of Na + for H + is shown to occur at the C-terminus and at up to all of the amide linkages using [Na 10 X 11 ] - , whereas only the C-terminus engages in Na + /H + exchange with [Na 10 Cl 11 ] - and [Na 10 (NO 3 ) 11 ] - . In the latter cases, an additional Na + is taken up as the excess positive charge, presumably due to solvation of the charge by multiple sites (e.g., carbonyl oxygens and basic sites).