Coherent diffractive imaging of proteins and viral capsids: simulating MS SPIDOC.

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm-1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

Protein Unfolding in Freeze Frames: Intermediate States are Revealed by Variable-Temperature Ion Mobility-Mass Spectrometry.

The gas phase is an idealized laboratory for the study of protein structure, from which it is possible to examine stable and transient forms of mass-selected ions in the absence of bulk solvent. With ion mobility-mass spectrometry (IM-MS) apparatus built to operate at both cryogenic and elevated temperatures, we have examined conformational transitions that occur to the monomeric proteins: ubiquitin, lysozyme, and α-synuclein as a function of temperature and in source activation. We rationalize the experimental observations with a temperature-dependent framework model and comparison to known conformers. Data from ubiquitin show unfolding transitions that proceed through diverse and highly elongated intermediate states, which converge to more compact structures. These findings contrast with data obtained from lysozyme─a protein where (un)-folding plasticity is restricted by four disulfide linkages, although this is alleviated in its reduced form. For structured proteins, collision activation of the protein ions in-source enables subsequent "freezing" or thermal annealing of unfolding intermediates, whereas disordered proteins restructure substantially at 250 K even without activation, indicating that cold denaturation can occur without solvent. These data are presented in the context of a toy model framework that describes the relative occupancy of the available conformational space.

Cold Denaturation of Proteins in the Absence of Solvent: Implications for Protein Storage.

The effect of temperature on the stability of proteins is well explored above 298 K, but harder to track experimentally below 273 K. Variable-temperature ion mobility mass spectrometry (VT IM-MS) allows us to measure the structure of molecules at sub-ambient temperatures. Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies (mAbs) on cooling by measuring their collision cross sections (CCS) at discrete drift gas temperatures from 295 to 160 K. The CCS at 250 K is larger than predicted from collisional theory and experimental data at 295 K. This restructure is attributed to change in the strength of stabilizing intermolecular interactions. Below 250 K the CCS of the mAbs increases in line with prediction implying no rearrangement. Comparing data from isotypes suggest disulfide bridging influences thermal structural rearrangement. These findings indicate that in vacuo deep-freezing minimizes denaturation and maintains the native fold and VT IM-MS measurements at sub ambient temperatures provide new insights to the phenomenon of cold denaturation.

Cold Denaturation of Proteins in the Absence of Solvent: Implications for Protein Storage.

The effect of temperature on the stability of proteins is well explored above 298 K, but harder to track experimentally below 273 K. Variable-temperature ion mobility mass spectrometry (VT IM-MS) allows us to measure the structure of molecules at sub-ambient temperatures. Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies (mAbs) on cooling by measuring their collision cross sections (CCS) at discrete drift gas temperatures from 295 to 160 K. The CCS at 250 K is larger than predicted from collisional theory and experimental data at 295 K. This restructure is attributed to change in the strength of stabilizing intermolecular interactions. Below 250 K the CCS of the mAbs increases in line with prediction implying no rearrangement. Comparing data from isotypes suggest disulfide bridging influences thermal structural rearrangement. These findings indicate that in vacuo deep-freezing minimizes denaturation and maintains the native fold and VT IM-MS measurements at sub ambient temperatures provide new insights to the phenomenon of cold denaturation.

Interplay between chromophore binding and domain assembly by the B12-dependent photoreceptor protein, CarH.

Organisms across the natural world respond to their environment through the action of photoreceptor proteins. The vitamin B12-dependent photoreceptor, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids to protect against photo-oxidative stress. The binding of B12 to CarH monomers in the dark results in the formation of a homo-tetramer that complexes with DNA; B12 photochemistry results in tetramer dissociation, releasing DNA for transcription. Although the details of the response of CarH to light are beginning to emerge, the biophysical mechanism of B12-binding in the dark and how this drives domain assembly is poorly understood. Here - using a combination of molecular dynamics simulations, native ion mobility mass spectrometry and time-resolved spectroscopy - we reveal a complex picture that varies depending on the availability of B12. When B12 is in excess, its binding drives structural changes in CarH monomers that result in the formation of head-to-tail dimers. The structural changes that accompany these steps mean that they are rate-limiting. The dimers then rapidly combine to form tetramers. Strikingly, when B12 is scarcer, as is likely in nature, tetramers with native-like structures can form without a B12 complement to each monomer, with only one apparently required per head-to-tail dimer. We thus show how a bulky chromophore such as B12 shapes protein/protein interactions and in turn function, and how a protein can adapt to a sub-optimal availability of resources. This nuanced picture should help guide the engineering of B12-dependent photoreceptors as light-activated tools for biomedical applications.

Coupling 193 nm Ultraviolet Photodissociation and Ion Mobility for Sequence Characterization of Conformationally-Selected Peptides.

Ultraviolet photodissociation (UVPD) has emerged as a useful technique for characterizing peptide, protein, and protein complex primary and secondary structure. 193 nm UVPD, specifically, enables extensive covalent fragmentation of the peptide backbone without the requirement of a specific side chain chromophore and with no precursor charge state dependence. We have modified a commercial quadrupole-ion mobility-time-of-flight (Q-IM-TOF) mass spectrometer to include 193 nm UVPD following ion mobility. Ion mobility (IM) is a gas-phase separation technique that enables separation of ions by their size, shape, and charge, providing an orthogonal dimension of separation to mass analysis. Following instrument modifications, we characterized the performance of, and information that could be generated from, this new setup using the model peptides substance P, melittin, and insulin chain B. These experiments show extensive fragmentation across the peptide backbone and a variety of ion types as expected from 193 nm UVPD. Additionally, y-2 ions (along with complementary a+2 and b+2 ions) N-terminal to proline were observed. Combining the IM separation and mobility gating capabilities with UVPD, we demonstrate the ability to accomplish both mass- and mobility-selection of bradykinin des-Arg9 and des-Arg1 peptides followed by complete sequence characterization by UVPD. The new capabilities of this modified instrument demonstrate the utility of combining IM with UVPD because isobaric species cannot be independently selected with a traditional quadrupole alone.

Coupling Droplet Microfluidics with Mass Spectrometry for Ultrahigh-Throughput Analysis of Complex Mixtures up to and above 30 Hz.

High- and ultrahigh-throughput label-free sample analysis is required by many applications, extending from environmental monitoring to drug discovery and industrial biotechnology. HTS methods predominantly are based on a targeted workflow, which can limit their scope. Mass spectrometry readily provides chemical identity and abundance for complex mixtures, and here, we use microdroplet generation microfluidics to supply picoliter aliquots for analysis at rates up to and including 33 Hz. This is demonstrated for small molecules, peptides, and proteins up to 66 kDa on three commercially available mass spectrometers from salty solutions to mimic cellular environments. Designs for chip-based interfaces that permit this coupling are presented, and the merits and challenges of these interfaces are discussed. On an Orbitrap platform droplet infusion rates of 6 Hz are used for analysis of cytochrome c, on a DTIMS Q-TOF similar rates were obtained, and on a TWIMS Q-TOF utilizing IM-MS software rates up to 33 Hz are demonstrated. The potential of this approach is demonstrated with proof of concept experiments on crude mixtures including egg white, unpurified recombinant protein, and a biotransformation supernatant.

Quantification of protein glycation using vibrational spectroscopy.

Glycation is a protein modification prevalent in the progression of diseases such as Diabetes and Alzheimer's, as well as a byproduct of therapeutic protein expression, notably for monoclonal antibodies (mAbs). Quantification of glycated protein is thus advantageous in both assessing the advancement of disease diagnosis and for quality control of protein therapeutics. Vibrational spectroscopy has been highlighted as a technique that can easily be modified for rapid analysis of the glycation state of proteins, and requires minimal sample preparation. Glycated samples of lysozyme and albumin were synthesised by incubation with 0.5 M glucose for 30 days. Here we show that both FTIR-ATR and Raman spectroscopy are able to distinguish between glycated and non-glycated proteins. Principal component analysis (PCA) was used to show separation between control and glycated samples. Loadings plots found specific peaks that accounted for the variation - notably a peak at 1027 cm-1 for FTIR-ATR. In Raman spectroscopy, PCA emphasised peaks at 1040 cm-1 and 1121 cm-1. Therefore, both FTIR-ATR and Raman spectroscopy found changes in peak intensities and wavenumbers within the sugar C-O/C-C/C-N region (1200-800 cm-1). For quantification of the level of glycation of lysozyme, partial least squares regression (PLSR), with statistical validation, was employed to analyse Raman spectra from solution samples containing 0-100% glycated lysozyme, generating a robust model with R2 of 0.99. We therefore show the scope and potential of Raman spectroscopy as a high throughput quantification method for glycated proteins in solution that could be applied in disease diagnostics, as well as therapeutic protein quality control.

Using Collision Cross Section Distributions to Assess the Distribution of Collision Cross Section Values.

Careful transfer of ions into the gas-phase permits the measurement of protein structures, with ion mobility-mass spectrometry, which provides shape and stoichiometry information. Collision cross sections (CCS) can be obtained from measurements made of the ions mobility through a given gas, and such structural information once obtained should also permit interlaboratory comparisons. However, until recently, there was not a recommended standard form for the reporting of such measurements. In this study, we explore the use of collision cross section distributions to allow comparisons of IM-MS data for commonly analyzed proteins. We present measurements from seven proteins across three IM-MS configurations, namely, an Agilent 6560 IMQToF, a Waters Synapt G2 possessing a TWIMS cell and a modified Synapt G2 possessing an RF confining linear field drift cell. Mobility measurements were taken using He and N2 as the drift gases. To aid comparability across instruments and best assess the corresponding gas-phase conformational landscapes of the protein "standards", we present the data in the form of averaged CCS distributions. For experiments carried out in N2, CCS values for the most compact ion conformations have an interinstrument variability of ≤3%, and the total CCS distributions are generally similar across platforms. For experiments carried out in He, we observe the total CCS distributions to follow the same trend as observed in N2, while CCS for the most compact ion conformations sampled on the 6560 are systematically smaller by up to 10% than those observed on the G2. The calibration procedure (for TWIMS) yields TWCCS for native-like proteins which are largely similar to those obtained on DTIMS instruments. We collate previously reported values of CCS for these proteins in the form of histograms which bear a remarkable similarity to the CCS distributions, reflecting the conformational heterogeneity of proteins and also how conformer populations can be altered on transfer from solution to the detector. This gives concern for some caution when calibrating sample protein drift times simply with single numeric CCS values.

The major secreted protein of the whipworm parasite tethers to matrix and inhibits interleukin-13 function.

Infection by soil transmitted parasitic helminths, such as Trichuris spp, are ubiquitous in humans and animals but the mechanisms determining persistence of chronic infections are poorly understood. Here we show that p43, the single most abundant protein in T. muris excretions/secretions, is non-immunogenic during infection and has an unusual sequence and structure containing subdomain homology to thrombospondin type 1 and interleukin (IL)-13 receptor (R) α2. Binding of p43 to IL-13, the key effector cytokine responsible for T. muris expulsion, inhibits IL-13 function both in vitro and in vivo. Tethering of p43 to matrix proteoglycans presents a bound source of p43 to facilitate interaction with IL-13, which may underpin chronic intestinal infection. Our results suggest that exploiting the biology of p43 may open up new approaches to modulating IL-13 function and control of Trichuris infections.

Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor.

UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light, resulting in dissociation into monomers, which are considered to be the active state and comprise a ß-propeller core domain and intrinsically disordered N- and C-terminal tails. The C terminus is required for functional binding to signaling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photoactivation. We show that, while truncated UVR8 photoconverts from a single conformation of dimers to a single monomer conformation, the full-length protein exists in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C terminus is primed for activation. In the monomer the extended C terminus destabilizes the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.

Initial Protein Unfolding Events in Ubiquitin, Cytochrome c and Myoglobin Are Revealed with the Use of 213 nm UVPD Coupled to IM-MS.

The initial stages of protein unfolding may reflect the stability of the entire fold and can also reveal which parts of a protein can be perturbed, without restructuring the rest. In this work, we couple UVPD with activated ion mobility mass spectrometry to measure how three model proteins start to unfold. Ubiquitin, cytochrome c and myoglobin ions produced via nESI from salty solutions are subjected to UV irradiation pre-mobility separation; experiments are conducted with a range of source conditions which alter the conformation of the precursor ion as shown by the drift time profiles. For all three proteins, the compact structures result in less fragmentation than more extended structures which emerge following progressive in-source activation. Cleavage sites are found to differ between conformational ensembles, for example, for the dominant charge state of cytochrome c [M + 7H]7+, cleavage at Phe10, Thr19 and Val20 was only observed in activating conditions whilst cleavage at Ala43 is dramatically enhanced. Mapping the photo-cleaved fragments onto crystallographic structures provides insight into the local structural changes that occur as protein unfolding progresses, which is coupled to global restructuring observed in the drift time profiles. Graphical Abstract.

The Use of Mass Spectrometry to Examine IDPs: Unique Insights and Caveats.

A sizeable proportion of active protein sequences lack structural motifs making them irresolvable by NMR and crystallography. Such intrinsically disordered proteins (IDPs) or regions (IDRs) play a major role in biological mechanisms. They are often involved in cell regulation processes, and by extension can be the perpetrator or signifier of disease. In light of their importance and the shortcomings of conventional methods of biophysical analysis to identify them and to describe their conformational variance, IDPs and IDRs have been termed "the dark proteome." In this chapter we describe the use of ion mobility-mass spectrometry (IM-MS) coupled with electrospray ionization to analyze the conformational diversity of IDPs. Using the LEA protein COR15A as an exemplar system and contrasting it with the behavior of myoglobin, we outline the methods for analyzing an IDP using nanoelectrospray ionization coupled with IM-MS, covering sample preparation, purification; optimization of mass spectrometry conditions and tuning parameters; data collection and analysis. Following this, we detail the use of a "toy" model that provides a predictive framework for the study of all proteins with ESI-IM-MS.

UV Photodissociation Mass Spectrometry Accurately Localize Sites of Backbone Deuteration in Peptides.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is now a routinely used technique to inform on protein structure, dynamics, and interactions. Localizing the incorporated deuterium content on a single residue basis increases the spatial resolution of this technique enabling detailed structural analysis. Here, we investigate the use of ultraviolet photodissociation (UVPD) at 213 nm to measure deuterium levels at single residue resolution in HDX-MS experiments. Using a selectively labeled peptide, we show that UVPD occurs without H/D scrambling as the peptide probe accurately retains its solution-phase deuterium labeling pattern. Our results indicate that UVPD provides an attractive alternative to electron mediated dissociation for increasing the spatial resolution of the HDX-MS experiment, capable of yielding high fragmentation efficiency, high fragment ion diversity, and low precursor ion charge-state dependency.

Cisplatin and transplatin interaction with methionine: bonding motifs assayed by vibrational spectroscopy in the isolated ionic complexes.

Cisplatin and transplatin (cis- and trans-[PtCl2(NH3)2]) have been allowed to react with methionine (Met) in water solution in a study aimed to characterize the monofunctional complex primarily formed. The thioether function of methionine is known to have a very high affinity for square planar platinum(ii) and sulfur-containing biomolecules have been proposed as a cisplatin drug reservoir on the way to platination at DNA. Both cisplatin and transplatin yield [PtCl(NH3)2Met]+ complexes, delivered by electrospray ionization in the gas phase and sampled as isolated species using tools based on mass spectrometry. The collision induced dissociation spectra of both cis-[PtCl(NH3)2Met]+ and trans-[PtCl(NH3)2Met]+ are quite similar and also the transport properties assayed by ion mobility mass spectrometry do not allow any appreciable discrimination. However, the vibrational spectra obtained by IR multiple photon absorption (IRMPD) spectroscopy show distinct features. Their analysis, supported by quantum chemical calculations, has revealed that while cisplatin attack is mainly directed to the sulfur atom of Met, transplatin shows a more balanced partition between sulfur and nitrogen binding. Among the vibrational signatures characterizing cis-[PtCl(NH3)2Met]+ and trans-[PtCl(NH3)2Met]+ complexes, the asymmetric NH2 stretching of the α-amino group of the amino acid at ca. 3440 cm-1 is peculiar and diagnostic of S-platination. IRMPD kinetics evaluated at this frequency support the prevailing S-attack by cisplatin while approximately a 1 : 2 ratio of S- versus N-coordination is observed by transplatin, to be possibly related to the trans effect at the platinum center.

Charge Mediated Compaction and Rearrangement of Gas-Phase Proteins: A Case Study Considering Two Proteins at Opposing Ends of the Structure-Disorder Continuum.

Charge reduction in the gas phase provides a direct means of manipulating protein charge state, and when coupled to ion mobility mass spectrometry (IM-MS), it is possible to monitor the effect of charge on protein conformation in the absence of solution. Use of the electron transfer reagent 1,3-dicyanobenzene, coupled with IM-MS, allows us to monitor the effect of charge reduction on the conformation of two proteins deliberately chosen from opposite sides of the order to disorder continuum: bovine pancreatic trypsin inhibitor (BPTI) and beta casein. The ordered BPTI presents compact conformers for each of three charge states accompanied by narrow collision cross-section distributions (TWCCSDN2→He). Upon reduction of BPTI, irrespective of precursor charge state, the TWCCSN2→He decreases to a similar distribution as found for the nESI generated ion of identical charge. The behavior of beta casein upon charge reduction is more complex. It presents over a wide charge state range (9-28), and intermediate charge states (13-18) have broad TWCCSDN2→He with multiple conformations, where both compaction and rearrangement are seen. Further, we see that the TWCCSDN2→He of the latter charge states are even affected by the presence of radical anions. Overall, we conclude that the flexible nature of some proteins result in broad conformational distributions comprised of many families, even for single charge states, and the barrier between different states can be easily overcome by an alteration of the net charge. Graphical Abstract ᅟ.

Use of Ultraviolet Photodissociation Coupled with Ion Mobility Mass Spectrometry To Determine Structure and Sequence from Drift Time Selected Peptides and Proteins.

We demonstrate the capabilities of a laser-coupled ion mobility mass spectrometer for analysis of peptide sequence and structure showing ultraviolet photodissociation (UVPD) spectra of mass and mobility selected ions. A Synapt G2-S mass spectrometer has been modified to allow photointeraction of ions post the mobility cell. For this work, we have employed a single wavelength laser, which irradiates at 266 nm. We present the unique capabilities of this instrument and demonstrate several key features. Irradiation of luteinizing hormone releasing hormone (LHRH), growth hormone releasing hexapeptide (GHRP-6), and TrpCage (sequence NLYIQWLKDGGPSSGRPPPS) yields extensive b- and y-type fragmentation as well as a- and c-type ions. In addition, we observe side chain losses, including the indole group from tryptophan, and immonium ions. For negatively charged ions, we show the advantage of using collision-induced dissociation (CID) post-UVPD: radical ions are produced following irradiation, and these fragment with higher efficiency. Further, we have incorporated ion mobility and subsequent drift time gating into the UVPD method allowing the separate analysis of m/z-coincident species, both conformers and multimers. To demonstrate, we selectively dissociate the singly charged dimer or doubly charged monomer of the peptide gramicidin A and conformers of the [M + 5H]5+ form of the peptide melittin. Each mobility selected form has a different "fingerprint" dissociation spectrum, both predominantly containing b and y fragments. Differences in the intensities of various loss channels between the two species were revealed. The smaller conformer of melittin has fewer cleavage sites along the peptide backbone than the larger conformer suggesting considerable structural differences. For gramicidin, a single laser shot UVPD discriminates between primary photodissociation and subsequent fragmentation of fragments. We also show how this modified instrument facilitates activated electron photodissociation. UVPD-IM-MS analysis serves both as a method for peptide sequencing for peptides of similar (or identical) m/z and a method for optical analysis of mobility separated species.

Epoxyqueuosine Reductase Structure Suggests a Mechanism for Cobalamin-dependent tRNA Modification.

Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5'-GUN-3' tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes.

The photochemical mechanism of a B12-dependent photoreceptor protein.

The coenzyme B12-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B12 the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.

Proton transfer in histidine-tryptophan heterodimers embedded in helium droplets.

We used cold helium droplets as nano-scale reactors to form and ionize, by electron bombardment and charge transfer, aromatic amino acid heterodimers of histidine with tryptophan, methyl-tryptophan, and indole. The molecular interaction occurring through an N-H···N hydrogen bond leads to a proton transfer from the indole group of tryptophan to the imidazole group of histidine in a radical cationic environment.