Project Summary Mass spectrometry and tandem mass spectrometry have become essential tools in virtually all of the molecular sciences associated with biomedical research. Many questions of interest can be addressed by breaking macromolecules of interest (e.g., proteins) into smaller fragments prior to analysis. However, information inherently present in intact macromolecules and their complexes is lost upon digestion, which has motivated the development of so-called “top-down proteomics” and “native mass spectrometry”. While major advances have been made in both of these areas, significant challenges remain, particularly for high mass heterogenous mixtures. This effort emphasizes novel ion chemistries and novel instrumentation directed to challenges in top- down tandem mass spectrometry of biopolymers and their complexes. Particular emphasis is placed on the attachment of multiply-charged ions to high-mass analyte ions of opposite polarity to facilitate mass measurement, mixture analysis, and structural characterization. The controlled attachment of reagents of known mass and charge can dramatically improve the ‘peak capacity’ of a mass spectrometry measurement applied to complex mixtures of ions derived from electrospray ionization. Furthermore, ion attachment, in conjunction with MSn workflows, may prove to be useful in revealing the surface exposure of components in a complex. Novel ion/ion reactions involving superacid anions may also prove to be useful as gas-phase means for the selective removal of metal ions that become incorporated in bio-complexes ionized under native conditions. This effort also involves instrument development aimed at supporting high mass-to-charge ion manipulation (e.g., digital ion trap operation for ion isolation and ion parking of high mass-to-charge ions) and measurement (digital ion trap operation and electrostatic ion trap mass analysis) to support multiply-charged ion attachment experiments. The effort will extend the utility of mass spectrometry and tandem mass spectrometry in top-down biopolymer characterization as well as in the mass measurement and characterization of large complexes, such as molecular machines and viruses.