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Mass Spectrometry-Based Approaches for the Analysis of Protein Higher Order Structure

Published: March 3, 2023 doi: 10.3791/64979


Higher order structure (HOS) is an important aspect of protein characterization as it is closely related to protein function. Mass spectrometry (MS) is one of the key methods for assessing protein HOS1. Although MS-based approaches are unable to resolve the protein structure with atomic resolution, they have the advantages of mid-to-high spatial resolution, high specificity, low sample amount requirements, high throughput, and proteomics capabilities.

MS-based protein footprinting approaches assess the protein HOS by analyzing the solvent-accessible surface of the protein, and they are usually coupled with different chemical labeling approaches. Reversible labeling, such as by hydrogen–deuterium exchange (HDX), targets the backbone amide hydrogens in the protein1,2. Irreversible labeling utilizes either chemicals that target specific amino acid residues3 or radicals that label a variety of different residues with higher reaction speed1. Chemical cross-linking is also part of the irreversible labeling category, yet its bivalent feature allows for the determination of spatial constraints between two cross-linked sites, which offers unique topological insights4,5. All these approaches can be combined to form a “toolbox”, making MS a valuable tool for elucidating protein HOS.

We organized this methods collection to present the recent advancements in the field of MS-based protein HOS analysis. The first set of articles feature HDX-based approaches. Habibi and Thibodeaux6 describe a relatively classical continuous, bottom-up HDX-MS approach. With in-solution hydrogen–deuterium (H/D) exchange at multiple exchange times followed by on-column pepsin digestion, the level of deuterium uptake can be determined at the peptide level. When performing differential experiments (i.e., bound versus unbound states), peptides that show either increased or decreased deuterium uptake levels represent areas that experience changes in HOS. The authors use lanthipeptide synthetases to nicely demonstrate the HDX-MS workflow.

Aside from the bottom-up approach, HDX-MS can also be performed in a top-down manner, allowing analysts to apply different separation strategies at the intact protein level. Chaihu et al.7 utilize capillary electrophoresis to separate different protein states or proteoforms that co-exist in the solution. Proteins undergo the H/D exchange during the electrophoresis while migrating in the capillary in the deuterated background electrolyte solution, and this is followed by top-down MS analysis and gas-phase fragmentations to assess the deuterium uptake levels at both the global and fragment levels. Lastly, there are also two other methods in this collection that highlight different methods of H/D exchange, namely exchange for lyophilized power8 and exchange after electrospray ionization9.

The second group of articles cover the irreversible labeling method. Among them, fast photochemical oxidation of proteins (FPOP) utilizes radicals as labeling species to mark the surface of the protein10,11. In practice, hydrogen peroxide is photoactivated through a laser to produce hydroxyl radicals, which serve as the active labeling species. Misra and Sharp12 present a strategy for radical dosimetry in which adenine is used as the reporter for the radical concentration and an in-line radical dosimetry system is used to monitor the adenine response. This allows the real-time compensation of the hydroxyl radical concentrations and, thus, the normalization of the reaction conditions to deliver an accurate readout. Subsequently, Weinberger and Chea collaborated with the Sharp group to develop a laser-free FPOP platform13. By replacing the hazardous laser with a plasma light source and combining this with the in-line radical dosimetry and an automated sample collecting system, they develop and provide an integrated solution for researchers who need to characterize protein HOS.

The three articles by Jones’ group14,15,16 explore the possibility of bringing FPOP and protein footprinting to in vivo studies. The advantage of this approach is unprecedented, as it allows the protein HOS to be analyzed in the native environment. The successful application of footprinting in cells14 and in Caenorhabditis elegans15 opens new possibilities for tracking the HOS of the whole proteome in its native state, thus allowing for a deep understanding of the protein HOS that cannot be achieved through other tools.

Another important method of irreversible labeling utilizes chemical reagents that target specific amino acid residues3. Kirsch et al.17 report a well-utilized workflow of protein footprinting through covalent labeling. Using diethylpyrocarbonate as the labeling reagent, the authors probe the changes in protein HOS by labeling a few kinds of amino acid residues on the surface of the protein. Through differential experiments followed by proteolytic digestion and bottom-up MS analysis, together the workflow allows for the identification of changes in solvent accessibilities, which directly relate to changes in HOS.

Lastly, chemical cross-linking is another important approach that utilizes bivalent chemical reagents to label proteins4,5. Chemical cross-linkers are used to cross-link two proteins that are spatially close to each other and, often,  form a structural complex. Haupt et al.18 report a protocol in which they demonstrate the use of chemical cross-linkers for understanding the architecture of protein complexes. The cross-linked protein complex can be analyzed at two different levels. Through proteolytic digestion followed by MS identification, the cross-linked dipeptides reveal the interacting sites of the protein complex. When analyzing the cross-linked sample through intact mass, the stoichiometric information on the protein subunits within the complex is readily available.

To summarize, we have witnessed substantial development in MS-based tools for protein HOS analysis over the past three decades. Irreversible and reversible footprinting coupled with MS analysis complement each other, and together they contribute significantly to protein HOS characterization. The works included in this methods collection represent stare-of-art methods in the MS-based protein HOS analysis field; these studies are a valuable resource for researchers who are trying to utilize MS-based approaches to answer critical questions. From a broader perspective, incorporating footprinting data into structural predictions is critical for breaking the limit of spatial resolution in MS-based methods. Pioneering works exist that demonstrate the feasibility of such an approach19,20. Additionally, platform integration is also crucial, as these approaches are complementary to each other and provide insights from different perspectives21,22. The methods reported in this collection demonstrate the power of such integration, and there will be continuous development in this direction. With all these advancements, the MS-based approach has grown beyond molecular weight determination, and continuous growth will be seen in this field in the coming decades.


The authors have nothing to disclose.


The authors have no acknowledgments.


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Mass Spectrometry Protein Higher Order Structure Hydrogen Deuterium Exchange Chemical Labeling Fast Photochemical Oxidation of Proteins Protein Chemical Cross-linking

Cite this Article

Liu, X. R., Huang, Y. MassMore

Liu, X. R., Huang, Y. Mass Spectrometry-Based Approaches for the Analysis of Protein Higher Order Structure. J. Vis. Exp. (193), e64979, doi:10.3791/64979 (2023).

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