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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.

SUBMITTER: Liu XR

PROVIDER: S-EPMC7531764 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The higher-order structure (HOS) of proteins plays a critical role in their function; therefore, it is important to our understanding of their function that we have as much information as possible about their three-dimensional structure and how it changes with time. Mass spectrometry (MS) has become an important tool for determining protein HOS owing to its high throughput, mid-to-high spatial resolution, low sample amount requirement and broad compatibility with various protein systems. Modern MS-based protein HOS analysis relies, in part, on footprinting, where a reagent reacts 'to mark' the solvent-accessible surface of the protein, and MS-enabled proteomic analysis locates the modifications to afford a footprint. Fast photochemical oxidation of proteins (FPOP), first introduced in 2005, has become a powerful approach for protein footprinting. Laser-induced hydrogen peroxide photolysis generates hydroxyl radicals that react with solvent-accessible side chains (14 out of 20 amino acid side chains) to fulfill the footprinting. The reaction takes place at sub-milliseconds, faster than most of labeling-induced protein conformational changes, thus enabling a 'snapshot' of protein HOS in solution. As a result, FPOP has been employed in solving several important problems, including mapping epitopes, following protein aggregation, locating small molecule binding, measuring ligand-binding affinity, monitoring protein folding and unfolding and determining hidden conformational changes invisible to other methods. Broader adoption will be promoted by dissemination of the technical details for assembling the FPOP platform and for dealing with the complexities of analyzing FPOP data. In this protocol, we describe the FPOP platform, the conditions for successful footprinting and its examination by mass measurements of the intact protein, the post-labeling sample handling and digestion, the liquid chromatography-tandem MS analysis of the digested sample and the data analysis with Protein Metrics Suite. This protocol is intended not only as a guide for investigators trying to establish an FPOP platform in their own lab but also for those willing to incorporate FPOP as an additional tool in addressing their questions of interest.

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

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