Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

Abstract

Proteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS determination of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS analysis, through which different covalent labeling approaches “mark” the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen–deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resolution, we present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biological questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a reference for investigators seeking a MS-based tool to address structural questions in protein science.

Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

“…Furthermore, the protocol for partial denaturation requires optimisation for each complex and can fail for proteins that are resistant to mild denaturants or precipitate easily upon denaturation. Other solution-phase methods exist to study higherorder protein structure by subsequent MS analysis, for example chemical crosslinking, 54 protein footprinting methods including fast photochemical oxidation of proteins (FPOP), 16 and hydrogen-deuterium exchange; [55][56][57][58] however, these are beyond the scope of this perspective. Integration of information from different native and non-native techniques can provide valuable structural insights.…”

“…12,13 The bottom-up approach has also been applied for higher-order structural characterisation using methods such as limited proteolysis, 14 chemical crosslinking, 15 and protein footprinting. 16 'Top-down' mass spectrometry, which forgoes the digestion step, has proven to be the premier MS-based technology for unambiguous proteoform characterisation, enabling in-depth sequencing, the discovery of novel proteoforms, and quantication of disease-associated PTMs. 13 While some technical Top-down proteomics; large-scale application of TDMS to (potentially) all proteins present in a cell, tissue, or organism, usually with the goal of understanding biological processes and gene expression control CID/CAD Collision-induced/collisionally activated dissociation; increasing the internal energy of ions by collisions with inert background gas molecules, a process in which energy is converted from translational to vibrational modes, resulting in dissociation of noncovalent and/or covalent bonds HCD Higher-energy collisional dissociation; used in Orbitrap instruments to distinguish 'beam-type' collisional activation in non-trapping multipoles from activation by resonant excitation in ion traps.…”

Accurate protein structure prediction with hydroxyl radical protein footprinting data

“…Covalent labeling involves exposure of a protein to a labeling reagent that will irreversibly modify residues. Residues that are more accessible to solvent are generally more likely to be covalently modified, providing insight into the tertiary protein structure 10 , 11 .…”

“…Covalent labeling can be achieved with a multitude of reagents, including carbenes, diethylpyrocarbonate, and hydroxyl radicals [11][12][13] . Hydroxyl radicals, a commonly used covalent labeling reagent, are frequently derived from radiolysis or photolysis of hydrogen peroxide or water 14,15 .…”

Self-Organized Amphiphiles are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments

“…Fast photochemical oxidation of proteins (FPOP), a method that generates hydroxyl radicals by UV laser-induced photolysis of hydrogen peroxide, has demonstrated the ability to react with a wide variety of amino acids on the surface of the proteins and label them without altering the native protein structure during labeling [7][8][9]. FPOP coupled with MS has been rapidly adopted to protein structural biology research in recent decades [7,[10][11][12]. However, FPOP coupled with MS for membrane protein analysis remains limited, in part due to the low extent of labeling that has been reported.…”