Brandon T. Ruotolo

Ion mobility–mass spectrometry analysis of large protein complexes

Here we describe a detailed protocol for both data collection and interpretation with respect to ion mobility–mass spectrometry analysis of large protein assemblies. Ion mobility is a technique that can separate gaseous ions based on their size and shape. Specifically, within this protocol, we cover general approaches to data interpretation, methods of predicting whether specific model structures for a given protein assembly can be separated by ion mobility, and generalized strategies for data normalization and modeling. The protocol also covers basic instrument settings and best practices for both observation and detection of large noncovalent protein complexes by ion mobility–mass spectrometry.

Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinsons disease and Alzheimers disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimers disease.

Collision Cross Sections of Proteins and Their Complexes: A Calibration Framework and Database for Gas-Phase Structural Biology

Collision cross sections in both helium and nitrogen gases were measured directly using a drift cell with RF ion confinement inserted within a quadrupole/ion mobility/time-of-flight hybrid mass spectrometer (Waters Synapt HDMS, Manchester, U.K.). Collision cross sections for a large set of denatured peptide, denatured protein, native-like protein, and native-like protein complex ions are reported here, forming a database of collision cross sections that spans over 2 orders of magnitude. The average effective density of the native-like ions is 0.6 g cm(-3), which is significantly lower than that for the solvent-excluded regions of proteins and suggests that these ions can retain significant memory of their solution-phase structures rather than collapse to globular structures. Because the measurements are acquired using an instrument that mimics the geometry of the commercial Synapt HDMS instrument, this database enables the determination of highly accurate collision cross sections from traveling-wave ion mobility data through the use of calibration standards with similar masses and mobilities. Errors in traveling-wave collision cross sections determined for native-like protein complexes calibrated using other native-like protein complexes are significantly less than those calibrated using denatured proteins. This database indicates that collision cross sections in both helium and nitrogen gases can be well-correlated for larger biomolecular ions, but non-correlated differences for smaller ions can be more significant. These results enable the generation of more accurate three-dimensional models of protein and other biomolecular complexes using gas-phase structural biology techniques.

Evidence for Macromolecular Protein Rings in the Absence of Bulk Water

We have examined the architecture of a protein complex in the absence of bulk water. By determining collision cross sections of assemblies of the trp RNA binding protein, TRAP, we established that the 11-membered ring topology of the complex can be maintained within a mass spectrometer. We also found that the binding of tryptophan enhances the stability of the ring structure and that addition of a specific RNA molecule increases the size of the complex and prevents structural collapse. These results provide definitive evidence that protein quaternary structure can be maintained in the absence of bulk water and highlight the potential of ion mobility separation for defining shapes of heterogeneous macromolecular assemblies.

Mass spectrometry: come of age for structural and dynamical biology.

Over the past two decades, mass spectrometry (MS) has emerged as a bone fide approach for structural biology. MS can inform on all levels of protein organization, and enables quantitative assessments of their intrinsic dynamics. The key advantages of MS are that it is a sensitive, high-resolution separation technique with wide applicability, and thereby allows the interrogation of transient protein assemblies in the context of complex mixtures. Here we describe how molecular-level information is derived from MS experiments, and how it can be combined with spatial and dynamical restraints obtained from other structural biology approaches to allow hybrid studies of protein architecture and movements.

Coupling Microdroplet Microreactors with Mass Spectrometry: Reading the Contents of Single Droplets Online

Fully integrated: Mass spectrometry has been integrated into a detection scheme for microdroplets that are created within microfluidic channels (see picture, scale bar 200 microm). This technique allows droplets to be identified based on the compounds they contain, and combines fluorescence screening with MS analysis. These experiments indicate how similar approaches can be applied to the ambitious goals of on-chip protein evolution and chemical synthesis.

Structural basis for the inhibition of activin signalling by follistatin

The secreted, multidomain protein follistatin binds activins with high affinity, inhibiting their receptor interaction. We have dissected follistatins domain structure and shown that the minimal activin‐inhibiting fragment of follistatin is comprised of the first and second Fs domains (Fs12). This protein can bind to activin dimer and form a stable complex containing two Fs12 molecules and one activin dimer. We have solved crystal structures of activin A alone and its complex with Fs12 fragment to 2 Å resolution. The complex structure shows how Fs12 molecules wrap around the back of the ‘wings’ of activin, blocking the type II receptor‐binding site on activin A. Arginine 192 in Fs2 is a key residue in this interaction, inserting itself in between activins fingers. Complex formation imposes a novel orientation for the EGF‐ and Kazal‐like subdomains in the Fs2 domain and activin A shows further variation from the canonical TGF‐β family fold. The structure provides a detailed description of the inhibitory mechanism and gives insights into interactions of follistatin with other TGF‐β family proteins.

Insights into antiamyloidogenic properties of the green tea extract (−)-epigallocatechin-3-gallate toward metal-associated amyloid-β species

Despite the significance of Alzheimer’s disease, the link between metal-associated amyloid-β (metal–Aβ) and disease etiology remains unclear. To elucidate this relationship, chemical tools capable of specifically targeting and modulating metal–Aβ species are necessary, along with a fundamental understanding of their mechanism at the molecular level. Herein, we investigated and compared the interactions and reactivities of the green tea extract, (−)-epigallocatechin-3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate; EGCG], with metal [Cu(II) and Zn(II)]–Aβ and metal-free Aβ species. We found that EGCG interacted with metal–Aβ species and formed small, unstructured Aβ aggregates more noticeably than in metal-free conditions in vitro. In addition, upon incubation with EGCG, the toxicity presented by metal-free Aβ and metal–Aβ was mitigated in living cells. To understand this reactivity at the molecular level, structural insights were obtained by ion mobility-mass spectrometry (IM-MS), 2D NMR spectroscopy, and computational methods. These studies indicated that (i) EGCG was bound to Aβ monomers and dimers, generating more compact peptide conformations than those from EGCG-untreated Aβ species; and (ii) ternary EGCG–metal–Aβ complexes were produced. Thus, we demonstrate the distinct antiamyloidogenic reactivity of EGCG toward metal–Aβ species with a structure-based mechanism.

Subunit architecture of intact protein complexes from mass spectrometry and homology modeling.

Proteomic studies have yielded detailed lists of protein components. Relatively little is known, however, of interactions between proteins or of their spatial arrangement. To bridge this gap, we are developing a mass spectrometry approach based on intact protein complexes. By studying intact complexes, we show that we are able to not only determine the stoichiometry of all subunits present but also deduce interaction maps and topological arrangements of subunits. To construct an interaction network, we use tandem mass spectrometry to define peripheral subunits and partial denaturation in solution to generate series of subcomplexes. These subcomplexes are subsequently assigned using tandem mass spectrometry. To facilitate this assignment process, we have developed an iterative search algorithm (SUMMIT) to both assign protein subcomplexes and generate protein interaction networks. This software package not only allows us to construct the subunit architecture of protein assemblies but also allows us to explore the limitations and potential of our approach. Using series of hypothetical complexes, generated at random from protein assemblies containing between six and fourteen subunits, we highlight the significance of tandem mass spectrometry for defining subunits present. We also demonstrate the importance of pairwise interactions and the optimal numbers of subcomplexes required to assign networks with up to fourteen subunits. To illustrate application of our approach, we describe the overall architecture of two endogenous protein assemblies isolated from yeast at natural expression levels, the 19S proteasome lid and the RNA exosome. In constructing our models, we did not consider previous electron microscopy images but rather deduced the subunit architecture from series of subcomplexes and our network algorithm. The results show that the proteasome lid complex consists of a bicluster with two tetrameric lobes. The exosome lid, by contrast, is a six-membered ring with three additional bridging subunits that confer stability to the ring and with a large subunit located at the base. Significantly, by combining data from MS and homology modeling, we were able to construct an atomic model of the yeast exosome. In summary, the architectural and atomic models of both protein complexes described here have been produced in advance of high-resolution structural data and as such provide an initial model for testing hypotheses and planning future experiments. In the case of the yeast exosome, the atomic model is validated by comparison with the atomic structure from X-ray diffraction of crystals of the reconstituted human exosome, which is homologous to that of the yeast. Overall therefore this mass spectrometry and homology modeling approach has given significant insight into the structure of two previously intractable protein complexes and as such has broad application in structural biology.

Ion mobility-mass spectrometry for structural proteomics.

Ion mobility coupled to mass spectrometry has been an important tool in the fields of chemical physics and analytical chemistry for decades, but its potential for interrogating the structure of proteins and multiprotein complexes has only recently begun to be realized. Today, ion mobility–mass spectrometry is often applied to the structural elucidation of protein assemblies that have failed high-throughput crystallization or NMR spectroscopy screens. Here, we highlight the technology, approaches and data that have led to this dramatic shift in use, including emerging trends such as the integration of ion mobility–mass spectrometry data with more classical (e.g., ‘bottom-up’) proteomics approaches for the rapid structural characterization of protein networks.