Integration of Mass Spectrometry Data for Structural Biology

Abstract

Mass spectrometry (MS) is increasingly being used to probe the structure and dynamics of proteins and the complexes they form with other macromolecules. There are now several specialised MS methods each with unique sample preparation, data acquisition and data processing protocols. Collectively these methods are referred to as structural MS and include, crosslinking-, hydrogen deuterium exchange-, hydroxyl radical footprinting-native, ion mobility-and top-down MS. Each of these provides a unique type of structural information, ranging from composition and stoichiometry through to residue level proximity and solvent accessibility. Structural MS has proved particularly beneficial in studying protein classes for which analysis by classic structural biology techniques proves challenging, such as glycosylated or intrinsically disordered proteins. To capture the structural details for a particular system, especially larger multiprotein complexes, more than one structural MS method with other structural and biophysical techniques is often required. Key to integrating these diverse data are computational strategies and software solutions to facilitate this process.We provide a background to the structural MS methods and briefly summarise other structural methods and how these are combined with MS. We then describe current state of the art approaches for the integration of structural MS data for structural biology. We quantify how often these methods are used together and provide examples where such combinations have been fruitful. To illustrate the power of integrative approaches, we discuss progress in solving the structures of the proteasome and the nuclear pore complex. We also discuss how information from structural MS, particularly pertaining to protein dynamics is not currently utilised in integrative workflows and how such information can provide a more accurate picture of the systems studied. We conclude by discussing new developments in the MS and computational fields that will further enable in-cell structural studies.

Challenges in describing the conformation and dynamics of proteins with ambiguous behavior

“…Overall, it remains very difficult to capture the dynamic properties of proteins; despite the availability of molecular dynamics simulations of increasing length, limited direct dynamics measurements from NMR and other structural biology approaches, and the observed conformational diversity in the PDB, the complexity of possible protein movements and their likelihood within the in vivo environment of proteins, in general, precludes the generation of relevant all-encompassing datasets. The increasing amount of data that indirectly indicates such behavior, from mass spectrometry proteomics ( Britt, Cragnolini, and Thalassinos, 2021 ) as well as from evolutionary and disease mutation sources, will be in this respect invaluable, as already indicated in our limited study. The challenge here lies in interconnecting the various diverse data sources and analyzing the resulting complex information, which is beyond direct human understanding and requires machine learning approaches, preferably interpretable so that concepts and first principles can be derived from them.…”

“…However, due to various experimental challenges, these methods have not become widely used in the community of structural biology. Valid future alternatives for both single proteins (folding) and in-cell determination of protein states might come from mass spectrometry-based methods such as cross-linking (XL-MS) or hydrogen–deuterium exchange (HDX-MS), which are becoming increasingly informative ( Britt, Cragnolini, and Thalassinos, 2021 ).…”

Generating Ensembles of Dynamic Misfolding Proteins

“…), but can also resolve species of the same mass, but different CCS (e.g., compact vs. expanded versions of isobaric species) ( Beveridge et al, 2019 ; Moons et al, 2020 ). The ability of native ESI-MS to detect small populations of protein conformers and separate them based on size (resolution of a few Da) and shape (CCS) has been powerful in the investigation of folding/misfolding and aggregation pathways ( Benesch et al, 2006 ; Smith et al, 2006 , 2007 ; Woods et al, 2013 ; Young et al, 2014 ; Britt et al, 2021 ) and in the assembly of dynamic chaperone assemblies ( Young et al, 2018 ). Theoretical models that allow the calculation of MS-derived restraints such as CCS are perhaps lacking, although significant progress in this area has been made recently ( Kulesza et al, 2018 ).…”

Probing heavy metal binding to phycobiliproteins

“…Here, we use native mass spectrometry (MS) to probe the mechanistic details behind how the phycobiliproteins, PC and APC, bind metal cations. Native MS allows protein and protein complexes to be analysed in their native-like state, preserving stoichiometry and binding equilibria between proteins and protein complexes, enabling us to determine how these are altered upon metal binding [20][21][22][23][24]. Phycobiliproteins have previously been successfully investigated by native MS showing that their native structure can be preserved [25][26][27].…”