Digital Ion Trap Isolation\nand Mass Analysis of Macromolecular\nAnalytes with Multiply Charged Ion Attachment

Multiply charged ions produced by electrospray ionization\n(ESI)\nof heterogeneous mixtures of macromolecular analytes under native\nconditions are typically confined to relatively narrow ranges of mass-to-charge\n(<i>m</i>/<i>z</i>) ratio, often with extensive\noverlap. This scenario makes charge and mass assignments extremely\nchallenging, particularly when individual charge states are unresolved.\nAn ion/ion reaction strategy involving multiply charged ion attachment\n(MIA) to the mixture components in a narrow range of <i>m</i>/<i>z</i> can facilitate charge and mass assignment. In\nMIA operation, multiply charged reagent ions are attached to the analyte\nions of opposite polarity to provide large <i>m</i>/<i>z</i> displacements resulting from both large changes in mass\nand charge. However, charge reduction of the high <i>m</i>/<i>z</i> ions initially generated under native ESI conditions\nrequires the ability to isolate high <i>m</i>/<i>z</i> ions and to analyze even higher <i>m</i>/<i>z</i> product ions. Digital ion trap (DIT) operation offers means for\nboth high <i>m</i>/<i>z</i> ion isolation and\nhigh <i>m</i>/<i>z</i> mass analysis, in addition\nto providing conditions for the reaction of oppositely charged ions.\nThe feasibility of conducting MIA experiments in a DIT that takes\nadvantage of high <i>m</i>/<i>z</i> ion operation\nis demonstrated here using a tandem 2D-3D DIT instrument. Proof-of-concept\nMIA experiments with cations derived from β-galactosidase using\nthe 20– charge state of human serum immunoglobulin G (IgG,\n∼149 kDa) as the reagent anion are described. MIA experiments\ninvolving mixtures of ions derived from the <i>E. coli.</i> ribosome are also described. For example, three components in a\nmixture of 70S particles (>2.2 MDa) were resolved and assigned\nwith\nmasses and charges following an MIA experiment involving the 20–\ncharge state of human serum IgG.