CIUSuite 2: Next-Generation Software for the Analysis of Gas-Phase Protein Unfolding Data.

Ion mobility-mass spectrometry (IM-MS) has become an important addition to the structural biology toolbox, but separating closely related protein conformations remain challenging. Collision-induced unfolding (CIU) has emerged as a valuable technique for distinguishing iso-cross-sectional protein and protein complex ions through their distinct unfolding pathways in the gas phase. The speed and sensitivity of CIU analyses, coupled with their information-rich data sets, have resulted in the rapid growth of CIU for applications, ranging from the structural assessment of protein complexes to the characterization of biotherapeutics. This growth has occurred despite a lag in the capabilities of informatics tools available to process the complex data sets generated by CIU experiments, resulting in laborious manual analysis remaining commonplace. Here, we present CIUSuite 2, a software suite designed to enable robust, automated analysis of CIU data across the complete range of current CIU applications and to support the implementation of CIU as a true high-throughput technique. CIUSuite 2 uses statistical fitting and modeling methods to reliably quantify features of interest within CIU data sets, particularly in data with poor signal quality that cannot be interpreted with existing analysis tools. By reducing the signal-to-noise requirements for handling CIU data, we are able to demonstrate reductions in acquisition time of up to 2 orders of magnitude over current workflows. CIUSuite 2 also provides the first automated system for classifying CIU fingerprints, enabling the next generation of ligand screening and structural analysis experiments to be accomplished in a high-throughput fashion.