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

Abstract

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-crosssectional 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.

Toward Automation of Collision-Induced Unfolding Experiments through Online Size Exclusion Chromatography Coupled to Native Mass Spectrometry

“…Chromatographic, IM and MS data were analyzed using MassLynx v4.1. CIU data were processed using the CIUSuite 2 v2.1 software 33 . Data sets were collected in triplicate (with three SEC runs for each replicate) to generate averaged CIU fingerprints.…”

Exploring the conformational landscape of a lanthipeptide synthetase using native mass spectrometry

“…These CIU experiments revealed a similar unfolding pattern for HalM2 and the HalM2:HalA2 complex, where the native conformations present under gentle activation conditions (trap collision energy < 40 V) unfold through a series of intermediates (40 V < trap collision energy < 70 V) to more highly denatured conformations (trap collision energy > 70 V). The CIU50 values determined from this data (which approximate the collision energy at which half of the protein is unfolded) [42][43][44] indicate a substantial stabilization of the HalM2 gas phase structure induced by HalA2 binding (ΔCIU +, **" = 8.5 ). Therefore, the non-covalent contacts between the peptide and synthetase appear to be extensive and are perhaps localized in labile regions of the enzyme that are more susceptible to unfolding in the gas phase.…”

“…and 56.0 V for HalM2 and HalM2:HalA2, respectively, using CIUSuite2 software as described in the Supporting Information. 44 CIU data were collected with the instrumental settings given in Table S6.…”

Native Ubiquitin Structural Changes Resulting from Complexation with β-methylamino-L-alanine (BMAA)

“…Entrance Grid Delta CIU plots. 34 Default parameters were used (i.e., no additional smoothing or processing).…”