Abstract
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 semiautomated 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 .
Highlights
Protein footprinting techniques are emerging as fast and reliable methods for investigating protein-protein or protein-small molecule interactions, which are central to biochemical processes
While the method provides promising results it suffers from back-exchange to hydrogen in the common protic solvents used in liquid chromatography-mass spectrometry (LC-Mass spectrometry (MS))[3] and H/D scrambling during collisional activation for MS/MS, which can make sub-peptide level analysis challenging.[4]
Protein footprinting provides a valuable tool for interrogating protein interactions, but improving the efficiency and reproducibility of analysis is essential for the field to grow
Summary
Protein footprinting techniques are emerging as fast and reliable methods for investigating protein-protein or protein-small molecule interactions, which are central to biochemical processes. In order to characterize these interactions, an array of high-resolution techniques are frequently used, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM), but these methods are time-consuming and require relatively large quantities of sample. One of the earliest and most widely used MS techniques is hydrogen-deuterium exchange (HDX), which probes solvent exposure of a protein’s surface through the uptake of deuterium.[2] HDX has been used to investigate both binding interactions and conformational dynamics. While the method provides promising results it suffers from back-exchange to hydrogen in the common protic solvents used in liquid chromatography-mass spectrometry (LC-MS)[3] and H/D scrambling during collisional activation for MS/MS, which can make sub-peptide level analysis challenging.[4]
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