Stabilising protein–protein interactions is challenging, yet therapeutically important. Native mass spectrometry can be used to monitor binding equilibria, allowing identification and measurement of novel protein–protein interaction stabilisers.

Covalent footprinting of proteins using reactive intermediates such as radicals and carbenes is emerging as a valuable tool for mapping surface accessibility, and hence binding sites of proteins. The approach generates a significant amount of mass spectrometry (MS) data, which can be time-consuming to process manually.PepFoot, a software package that allows semi-automated processing of MS data from footprinting experiments, is described. By using the open source .mz5 file format, it is able to accept data from all the major instrument manufacturers. Following manual user interrogation of one data file within a user-friendly GUI, the software then automates determination of the degree of fractional modification (fm) with the footprinting agent across a batch of experimental data. This greatly increases efficiency and throughput compared to manual analysis of each file, and provides initial scrutiny and confidence compared to fully-automated analysis. Histogram plots of fm for each peptide from the footprinted protein may be displayed within PepFoot and mapped onto an imported protein structure to reveal differential labeling patterns and hence binding sites. The software has been tested on data from carbene and hydroxyl radical labeling experiments to demonstrate its broad utility.PepFoot is released under the LGPL version 3 license, and is available for Windows, MacOS and Linux systems at github.com/jbellamycarter/pepfoot.

Native mass spectrometry is a widely used tool in structural biology, providing information on protein structure and interactions through preservation of complexes in the gas phase. Herein, the importance of intramolecular non‐covalent interactions in the gas phase has been studied by alanine scanning and collision‐induced unfolding (CIU) ion mobility‐mass spectrometry. Mutation of specific polar and ionic residues on the surface of an acyl carrier protein (ACP) were found to destabilise the compact gas‐phase structure with mutants E31A, D32A, D41A and D65A being particularly destabilised. Molecular dynamics simulations of the ACP 7+ and 8+ ions showed extended intramolecular interactions, resulting from sidechain collapse of polar surface residues, which were confined to the gas phase and consistent with the CIU data. These findings provide evidence for the importance of specific ionic residues, and their interactions, in the maintenance of compact protein gas‐phase structure.

Native mass spectrometry is now an important tool in structural biology. Thus, the nature of higher protein structure in the vacuum of the mass spectrometer is an area of significant interest. One of the major goals in the study of gas‐phase protein structure is to elucidate the stabilising role of interactions at the level of individual amino acid residues. A strategy combining protein chemical modification together with collision induced unfolding (CIU) was developed and employed to probe the structure of compact protein ions produced by native electrospray ionisation. Tractable chemical modification was used to alter the properties of amino acid residues, and ion mobility‐mass spectrometry (IM‐MS) utilised to monitor the extent of unfolding as a function of modification. From these data the importance of specific intramolecular interactions for the stability of compact gas‐phase protein structure can be inferred. Using this approach, and aided by molecular dynamics simulations, an important stabilising interaction between K6 and H68 in the protein ubiquitin was identified, as was a contact between the N‐terminus and E22 in a ubiquitin binding protein UBA2.

Cyanobacteria, also known as blue–green algae, are ubiquitous organisms on the planet. They contain tremendous protein machineries that are of interest to the biotechnology industry and beyond. Recently, the number of annotated cyanobacterial genomes has expanded, enabling structural studies on known gene-coded proteins to accelerate. This review focuses on the advances in mass spectrometry (MS) that have enabled structural proteomics studies to be performed on the proteins and protein complexes within cyanobacteria. The review also showcases examples whereby MS has revealed critical mechanistic information behind how these remarkable machines within cyanobacteria function.