The diverse and expanding role of mass spectrometry in structural and molecular biology

Abstract

The emergence of proteomics has led to major technological advances in mass spectrometry (MS). These advancements not only benefitted MS-based high-throughput proteomics but also increased the impact of mass spectrometry on the field of structural and molecular biology. Here, we review how state-of-the-art MS methods, including native MS, top-down protein sequencing, cross-linking-MS, and hydrogen-deuterium exchange-MS, nowadays enable the characterization of biomolecular structures, functions, and interactions. In particular, we focus on the role of mass spectrometry in integrated structural and molecular biology investigations of biological macromolecular complexes and cellular machineries, highlighting work on CRISPR-Cas systems and eukaryotic transcription complexes.

Keywords: CRISPR–Cas; RNA polymerase; ligand binding; post‐translational modifications; protein complexes.

Figure 1. MS‐based molecular and structural biology reaches out over all cellular compartments and kingdoms of life

Shown are exemplary results of studies that employed biomolecular MS techniques. Clockwise (from bottom left): membrane interactions of the endosomal Vps34 complex (Rostislavleva et al, 2015), conformational dynamics of the ABC transporter P‐glycoprotein (Marcoux et al, 2013), the organization of the nuclear pore complex scaffold (Bui et al, 2013), nucleotide binding to the p97 ATPase (Schuller et al, 2016), the interfaces of the H2A‐H2B‐Nap1 histone‐chaperone complex (D'Arcy et al, 2013), the structure of the 55S mitochondrial ribosome (Greber et al, 2015), the norovirus assembly pathway (Uetrecht et al, 2010a), the exosome complex topology (Hernández et al, 2006; Synowsky et al, 2006; Shi et al, 2015), and the architecture of the dynein cofactor dynactin (Urnavicius et al, 2015). Structural snapshots adapted from the referenced publications with permission.

Figure 2. The broad scope of complementary biomolecular MS methods

In protein‐centric approaches (top), non‐digested biomolecules or biomolecular assemblies are analyzed. Typically, the sample is injected into the mass spectrometer via electrospray ionization using a conductive borosilicate capillary, as symbolized in the top panel. After injection, the analyte structure and composition can be further evaluated by several gas‐phase manipulation strategies, some of which are schematically depicted. Ion mobility separation prior to mass measurement renders information about the analyte's conformation and shape. Dissociation of a non‐denatured biomolecular assembly by collisional activation (symbolized as an explosion cartoon) allows inferring its stoichiometry and topological aspects. Mass selection can be performed for both denatured and non‐denatured analytes, enabling the focused analysis of a specific species such as a protein isoform that carries a certain mutation or a defined number of PTMs. After mass selection, proteoform sequencing can be performed by fragmenting the protein isoforms in the gas phase (symbolized as an explosion cartoon), which allows to determine their amino acid sequence and thus locate mutated and/or modified residues. In peptide‐centric approaches (bottom), proteins or protein complexes are manipulated in solution by traditional biochemical methods, such as chemical cross‐linking, limited proteolysis, or surface labeling. Subsequently, the proteins are digested into peptides and liquid chromatography/tandem‐MS is used as a highly accurate method to detect and identify the resulting peptides. A combination of the depicted peptide‐ and protein‐centric approaches, as shown in the center, gives insights into a multitude of biochemical and structural properties of the studied protein assemblies.

Figure 3. Basic principles of biomolecular MS

(A) Essential components of a mass spectrometer. The ion source facilitates the transfer of the analyte into the gas phase. The low‐resolution mass analyzer enables mass selection of specific analyte ion species and, in case of ion traps, may also be utilized for ion activation/fragmentation. Otherwise, gas‐phase fragmentation, for example, using CID, HCD, or ETD, takes place in the collision cell. Finally, a high‐resolution FTICR, TOF, or Orbitrap mass analyzer facilitates precise and accurate mass measurements. (B) Schematic representation of ESI and MALDI, the most commonly used ionization techniques in biomolecular MS. ESI produces multiply charged analyte ions (shown in yellow, orange, red, and purple) directly from a sample solution. In MALDI, a laser is used to ablate a mixture of matrix (shown in blue) and analyte molecules from a metal plate into the mass spectrometer, yielding predominantly singly charged ions. (C) Nomenclature of peptide fragment ions according to Roepstorff and Fohlman (1984) and Biemann (1990).

Figure 4. Complementary biomolecular MS data exemplified using the human hemoglobin model system

(A) Schematic of native holo‐hemoglobin and its constituents: two α‐chains, two β‐chains, and 4 heme groups. (B) Cross‐linking‐MS. The displayed MS2 spectrum represents two linked peptides derived from cross‐linked holo‐hemoglobin. The ion signals, shown as blue and red sticks, correspond to specific fragments of the cross‐linked peptides. These fragment ions enable the sequencing of both peptides and the localization of their linkage site, as indicated by the fragment ion map in the inset. Since two residues are only cross‐linked if they are in close spatial proximity, the identified cross‐link gives insights into the in‐solution structure of holo‐hemoglobin. (C) HDX‐MS. Shown are the isotope distributions of the same peptide derived from either holo‐hemoglobin (left panel) or the free hemoglobin α‐chain (right panel) after they were separately incubated in D₂O‐containing buffer. The incubations were quenched at three different time points. The isotope distribution remains at the same m/z position, when the peptide is derived from holo‐hemoglobin, whereas it gradually moves to higher m/z, when the peptide is derived from the free α‐chain. This shows that only in the free α‐chain, the peptide is able to take up the heavy deuterium isotope. Consequently, the peptide is solvent accessible in the free α‐chain, whereas it is solvent protected in holo‐hemoglobin. (D) Protein‐centric MS. Mass spectra of non‐digested hemoglobin were acquired under denaturing (upper mass spectrum) and native (lower mass spectrum) conditions. Under denaturing conditions, all signals are concentrated in the low m/z region since the ions of the unfolded proteins are highly charged. The inset illustrates that the heme cofactor is present as a singly charged ion (brown), whereas the hemoglobin α‐chain (red) and β‐chain (blue) are detected in several charge states. From these charge state envelopes, their accurate molecular masses can be derived (α‐chain = 15,155 ± 1 Da, β‐chain = 15,895 ± 1 Da, heme = 616.5 Da). In the native mass spectrum, the signals are shifted to higher m/z, indicating that hemoglobin is detected in its folded state (see main text). The molecular weight can be calculated as 64,588 ± 41 Da. Together with the constituent masses derived from the denaturing MS experiment, this result unambiguously evidences that holo‐hemoglobin is an α₂β₂ heterotetramer with four bound heme ligands.

Figure 5. Hybrid approaches gradually unravel the Mediator complex architecture

The Mediator complex is a challenging target for structural biology because it comprises several subcomplexes and highly flexible regions. First, a topological model of the Mediator middle module was derived with a hybrid approach employing native MS and IMS‐MS. This could be extended to a more detailed model by means of cross‐linking‐MS and homology modeling. In parallel, a Mediator head module structure was derived by combining X‐ray crystallography and cross‐linking‐MS results. Finally, hybrid structural biology approaches led to the characterization of a 15‐subunit Mediator core complex and a 21‐subunit Mediator complex. Structural images adapted with permission from the publications referenced in the figure.