SLIMPHONY: A SLIM-Based Instrument That Orchestrates Complex Ion Mobility-Mass Spectrometry Experiments.

The inherent heterogeneity of biological macromolecules offers a unique challenge for analysis. The combination of ion mobility (IM) and mass spectrometry (MS) is sensitive to the size, shape, and dynamics of, for example, proteins and their complexes. Combining multiple dimensions of ion mobility and mass spectrometry (IM-IM-MS) while leveraging unique gas-phase manipulations between dimensions has great potential for increasing the information content for challenging analytes. Here, we introduce an instrument, SLIMPHONY, which was built using the Structures for Lossless Ion Manipulations (SLIM) architecture. SLIMPHONY is unique in that eight independently controlled traveling-wave regions work in concert to enable complex, multidimensional separations. Single-dimension IM-MS experiments were used to separate a mixture of protein and protein-complex ions and demonstrate that the peak-to-peak resolution increases roughly with the square root of the separation length for a pair of hexakis(fluoroalkoxy)phosphazine ions. Ion selection and trapping between dimensions was then used to probe the gas-phase unfolding of a subpopulation of ubiquitin ions. Finally, by varying the guard potential used to confine ions, we demonstrate tunable activation of ubiquitin subpopulations, which we analyzed using IM separations of various lengths. With the ability to select and activate ions in multiple regions, to vary the number of dimensions of IM, and to control the length of IM separation, SLIMPHONY is a flexible platform for characterizing protein ions.