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Understanding the relationship between protein structure and experimental data is crucial for utilizing experiments to solve biochemical problems and optimizing the use of sparse experimental data for structural interpretation. Tandem mass spectrometry (MS/MS) can be used with a variety of methods to collect structural data for proteins. One example is surface-induced dissociation (SID), which is used to break apart protein complexes (via a surface collision) into intact subcomplexes and can be performed at multiple laboratory frame SID collision energies. These energy-resolved MS/MS experiments have shown that the profile of the breakages depends on the acceleration energy of the collision. It is possible to extract an appearance energy (AE) from energy-resolved mass spectrometry (ERMS) data, which shows the relative intensity of each type of subcomplex as a function of SID acceleration energy. We previously determined that these AE values for specific interfaces correlated with structural features related to interface strength. In this study, we further examined the structural relationships by developing a method to predict the full ERMS plot from the structure, rather than extracting a single value. First, we noted that for proteins with multiple interface types, we could reproduce the correct shapes of breakdown curves, further confirming previous structural hypotheses. Next, we demonstrated that interface size and energy density (measured using Rosetta) correlated with data derived from the ERMS plot (

Native mass spectrometry (nMS) is evolving into a workhorse for structural biology. The plethora of online and offline preparation, separation, and purification methods as well as numerous ionization techniques combined with powerful new hybrid ion mobility and mass spectrometry systems has established the great potential of nMS as a workhorse for structural biology. Fundamental to the progression of nMS has been the development of novel activation methods for dissociating proteins and protein complexes to deduce primary, secondary, tertiary, and quaternary structure through the combined use of multiple MS/MS technologies. This review highlights the key features and advantages of surface collisions (surface-induced dissociation, SID) for probing the connectivity of subunits within protein and nucleoprotein complexes and, in particular, for solving protein structure in conjunction with complementary techniques such as cryo-EM and computational modeling. A focus on several case studies wherein SID provided connectivity maps that were otherwise inaccessible by ‘gold standard’ structural biology techniques, or that agreed with solved crystal or cryo-EM structures, highlights the significant role SID, and more generally nMS, will play in structural elucidation of biological assemblies in the future as the technology becomes more widely adopted.

Native mass spectrometry (nMS) enables intact non-covalent complexes to be studied in the gas phase. nMS can provide information on composition, stoichiometry, topology, and, when coupled with surface-induced dissociation (SID), subunit connectivity. Here we describe the characterization of protein complexes by nMS and SID. Substructural information obtained using this method is consistent with the solved complex structure, when a structure exists. This provides confidence that the method can also be used to obtain substructural information for unknowns, providing insight into subunit connectivity and arrangements. High-energy SID can also provide information on proteoforms present. Previously SID has been limited to a few in-house modified instruments and here we focus on SID implemented within an in-house-modified Q Exactive UHMR. However, SID is currently commercially available within the Waters Select Series Cyclic IMS instrument. Projects are underway that involve the NIH-funded native MS resource (nativems.osu.edu), instrument vendors, and third-party vendors, with the hope of bringing the technology to more platforms and labs in the near future. Currently, nMS resource staff can perform SID experiments for interested research groups.

Chemical science 13(39), 11680 - 11695 (2022). doi:10.1039/D2SC02794A, Over half the proteins in the E. coli cytoplasm form homo or hetero-oligomeric structures. Experimentally determined structures are often considered in determining a protein's oligomeric state, but static structures miss the dynamic equilibrium between different quaternary forms. The problem is exacerbated in homo-oligomers, where the oligomeric states are challenging to characterize. Here, we re-evaluated the oligomeric state of 17 different bacterial proteins across a broad range of protein concentrations and solutions by native mass spectrometry (MS), mass photometry (MP), size exclusion chromatography (SEC), and small-angle X-ray scattering (SAXS), finding that most exhibit several oligomeric states. Surprisingly, some proteins did not show mass-action driven equilibrium between the oligomeric states. For approximately half the proteins, the predicted oligomeric forms described in publicly available databases underestimated the complexity of protein quaternary structures in solution. Conversely, AlphaFold multimer provided an accurate description of the potential multimeric states for most proteins, suggesting that it could help resolve uncertainties on the solution state of many proteins., Published by RSC, Cambridge

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.

Native mass spectrometry (nMS) has emerged as an important tool in studying the structure and function of macromolecules and their complexes in the gas phase. In this review, we cover recent advances in nMS and related techniques including sample preparation, instrumentation, activation methods, and data analysis software. These advances have enabled nMS-based techniques to address a variety of challenging questions in structural biology. The second half of this review highlights recent applications of these technologies and surveys the classes of complexes that can be studied with nMS. Complementarity of nMS to existing structural biology techniques and current challenges in nMS are also addressed.

In the study of membrane proteins and antimicrobial peptides, nanodiscs have emerged as a valuable membrane mimetic to solubilze these molecules in a lipid bilayer. We present the structural characterization of nanodiscs using native mass spectrometry and surface-induced dissociation, which are powerful tools in structural biology.

A variety of techniques involving the use of mass spectrometry (MS) have been developed to obtain structural information on proteins and protein complexes. One example of these techniques, surface-induced dissociation (SID), has been used to study the oligomeric state and connectivity of protein complexes. Recently, we demonstrated that appearance energies (AE) could be extracted from SID experiments and that they correlate with structural features of specific protein-protein interfaces. While SID AE provides some structural information, the AE data alone are not sufficient to determine the structures of the complexes. For this reason, we sought to supplement the data with computational modeling, through protein-protein docking. In a previous study, we demonstrated that the scoring of structures generated from protein-protein docking could be improved with the inclusion of SID data; however, this work relied on knowledge of the correct tertiary structure and only built full complexes for a few cases. Here, we performed docking using input structures that require less prior knowledge, using homology models, unbound crystal structures, and bound+perturbed crystal structures. Using flexible ensemble docking (to build primarily subcomplexes from an ensemble of backbone structures), the RMSD100 of all (15/15) predicted structures using the combined Rosetta, cryo-electron microscopy (cryo-EM), and SID score was less than 4 A, compared to only 7/15 without SID and cryo-EM. Symmetric docking (which used symmetry to build full complexes) resulted in predicted structures with RMSD100 less than 4 A for 14/15 cases with experimental data, compared to only 5/15 without SID and cryo-EM. Finally, we also developed a confidence metric for which all (26/26) proteins flagged as high confidence were accurately predicted.

Characterizing protein-protein interactions, stoichiometries, and subunit connectivity is key to understanding how subunits assemble into biologically-relevant, multi-subunit protein complexes. Native mass spectrometry (nMS) has emerged as a powerful tool to study protein complexes due to its low sample consumption and tolerance for heterogeneity. In nMS, positive mode ionization is routinely used and charge reduction, through the addition of solution additives, is often used, as the resulting lower charge states are often more compact and considered more native-like. When fragmented by surface-induced dissociation (SID), charge reduced complexes often give increased structural information over their “normal-charged” counter parts. A disadvantage of solution phase charge-reduction is that increased adduction, and hence peak broadening, is often observed. Previous studies have shown that protein complexes ionized using negative mode generally form lower charge states relative to positive mode. Here we demonstrate that the lower charged protein complex anions activated by SID fragment in a manner consistent with their solved structures, hence providing substructural information. Negative mode ionization in ammonium acetate offers the advantage of charge reduction without the peak broadening associated with solution phase charge reduction additives and provides direct structural information, when coupled with SID. SID of 20S human proteasome (a 28-mer comprised of four stacked heptamer rings in an αββα formation), for example, provides information on both substructure (e.g., splitting into a 7α ring and the corresponding ββα 21-mer; and into α dimers and trimers to provide connectivity around the 7 α ring) and proteoform information on monomers.

Building a barrel Computational design offers the possibility of making proteins with customized structures and functions. The range of accessible protein scaffolds has expanded with the design of increasingly complex cytoplasmic proteins and, recently, helical membrane proteins. Vorobieva et al. describe the successful computational design of eight-stranded transmembrane β-barrel proteins (TMBs). Using an iterative approach, they show the importance of negative design to prevent off-target structures and gain insight into the sequence determinants of TMB folding. Twenty-three designs satisfied biochemical screens for a TMB structure, and two structures were experimentally validated by nuclear magnetic resonance spectroscopy or x-ray crystallography. This is a step toward the custom design of pores for applications such as single-molecule sequencing. Science , this issue p. eabc8182

Iron-sulfur (Fe-S) cluster biosynthesis involves the action of a variety of functionally distinct proteins, most of which are evolutionarily conserved. Mutations in these Fe-S scaffold and trafficking proteins can cause diseases such as multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalopathy. Herein, we investigate the effect of Ile67Asn substitution in the BOLA3 protein that results in the MMDS2 phenotype. Although the exact functional role of BOLA3 in Fe-S cluster biosynthesis is not known, the [2Fe-2S]-bridged complex of BOLA3 with GLRX5, another Fe-S protein, has been proposed as a viable intermediary cluster carrier to downstream targets. Our investigations reveal that the Ile67Asn substitution impairs the ability of BOLA3 to bind its physiological partner GLRX5, resulting in a failure to form the [2Fe-2S]-bridged complex. Although no drastic structural change in BOLA3 arises from the substitution, as evidenced by wild-type and mutant BOLA3 1H-15N HSQC and ion mobility native mass spectrometry experiments, this substitution appears to influence cluster reconstitution on downstream proteins leading to the disease phenotype. By contrast, substituted derivatives of the holo homodimeric form of BOLA3 are formed and remain active toward cluster exchange.

The ability of naturally occurring transmembrane β-barrel proteins (TMBs) to spontaneously insert into lipid bilayers and form stable transmembrane pores is a remarkable feat of protein evolution and has been exploited in biotechnology for applications ranging from single molecule DNA and protein sequencing to biomimetic filtration membranes. Because it has not been possible to design TMBs from first principles, these efforts have relied on re-engineering of naturally occurring TMBs that generally have a biological function very different from that desired. Here we leverage the power of de novo computational design coupled with a “hypothesis, design and test” approach to determine principles underlying TMB structure and folding, and find that, unlike almost all other classes of protein, locally destabilizing sequences in both the β-turns and β-strands facilitate TMB expression and global folding by modulating the kinetics of folding and the competition between soluble misfolding and proper folding into the lipid bilayer. We use these principles to design new eight stranded TMBs with sequences unrelated to any known TMB and show that they insert and fold into detergent micelles and synthetic lipid membranes. The designed proteins fold more rapidly and reversibly in lipid membranes than the TMB domain of the model native protein OmpA, and high resolution NMR and X-ray crystal structures of one of the designs are very close to the computational model. The ability to design TMBs from first principles opens the door to custom design of TMBs for biotechnology and demonstrates the value of de novo design to investigate basic protein folding problems that are otherwise hidden by evolutionary history.One sentence summarySuccess in de novo design of transmembrane β-barrels reveals geometric and sequence constraints on the fold and paves the way to design of custom pores for sequencing and other single-molecule analytical applications.

Many mass spectrometry applications make use of tandem mass spectrometry, where two stages of m/z analysis are coupled. In between the two stages of m/z analysis, an activation or reaction step is carried out to cause either structurally-informative fragmentation or structurally-characteristic reaction of the precursor ion of interest. This review focuses on the use of collisions with a surface (surface-induced dissociation, SID) as the activation method in tandem mass spectrometry, with an emphasis on SID papers published over the past four years. SID is described and compared with other activation methods. The major application focused on in this review is the structural characterization of native protein complexes, complexes kinetically trapped that retain native-like solution structures upon transfer to the gas-phase and throughout the relatively short timeframe of the mass spectrometry experiment. Other SID applications currently under investigation are also briefly described. Pioneering work on SID ...

Proliferating cell nuclear antigen (PCNA) is a trimeric ring-shaped clamp protein that encircles DNA and interacts with many proteins involved in DNA replication and repair. Despite extensive structural work to characterize the monomeric, dimeric, and trimeric forms of PCNA alone and in complex with interacting proteins, no structure of PCNA in a ring-open conformation has been published. Here, we use a multidisciplinary approach, including single-molecule Förster resonance energy transfer (smFRET), native ion mobility-mass spectrometry (IM-MS), and structure-based computational modeling, to explore the conformational dynamics of a model PCNA from Sulfolobus solfataricus (Sso), an archaeon. We found that Sso PCNA samples ring-open and ring-closed conformations even in the absence of its clamp loader complex, replication factor C, and transition to the ring-open conformation is modulated by the ionic strength of the solution. The IM-MS results corroborate the smFRET findings suggesting that PCNA dynamics are maintained in the gas phase and further establishing IM-MS as a reliable strategy to investigate macromolecular motions. Our molecular dynamic simulations agree with the experimental data and reveal that ring-open PCNA often adopts an out-of-plane left-hand geometry. Collectively, these results implore future studies to define the roles of PCNA dynamics in DNA loading and other PCNA-mediated interactions.

Surface-induced dissociation (SID) was first introduced as an ion activation method for tandem mass spectrometry in the laboratory of Graham Cooks at Purdue University. Early studies focused on small molecules that could be ionized and fragmented by the instruments available at that time. It was predicted that SID would someday be applied to much more massive ions and that the large collision target (the surface) would be advantageous for dissociation of very large ions. This chapter describes the development of SID from its early days until the present, closing with examples that illustrate the benefits of using SID to cleave large noncovalent protein complexes.

To fulfill their biological functions, proteins must interact with their specific binding partners and often function as large assemblies composed of multiple proteins or proteins plus other biomolecules. Structural characterization of these complexes, including identification of all binding partners, their relative binding affinities, and complex topology, is integral for understanding function. Understanding how proteins assemble and how subunits in a complex interact is a cornerstone of structural biology. Here we report a native mass spectrometry (MS)-based method to characterize subunit interactions in globular protein complexes. We demonstrate that dissociation of protein complexes by surface collisions, at the lower end of the typical surface-induced dissociation (SID) collision energy range, consistently cleaves the weakest protein:protein interfaces, producing products that are reflective of the known structure. We present here combined results for multiple complexes as a training set, two validation cases, and four computational models. We show that SID appearance energies can be predicted from structures via a computationally derived expression containing three terms (number of residues in a given interface, unsatisfied hydrogen bonds, and a rigidity factor).

Native ion mobility mass spectrometry (MS) and surface induced dissociation (SID) are applied to study the integral membrane protein complexes AmtB and AqpZ. Fragments produced from SID are consistent with the solved structures of these complexes. SID is, therefore, a promising tool for characterization of membrane protein complexes.

This communication reports the identification of gas phase isomers in monolayer-protected silver clusters. Two different isomers of Ag11(SG)7(-) (SG-gulathione thiolate) with different drift times have been detected using combined electrospray ionization (ESI) and ion mobility (IM) mass spectrometry (MS). Surface induced dissociation (SID) of the 3(-) charge state of such clusters shows charge stripping to give the 1(-) charged ion with some sodium attachment, in addition to fragmentation. SID and collision induced dissociation (CID) for Ag11(SG)7(-) suggest different pathways being accessed with each method. SID was introduced for the first time for the study of monolayer-protected clusters.

Ion mobility mass spectrometry can be combined with data from top-down sequencing to discern adopted conformations of proteins in the absence of solvent. This multi-technique approach has particular applicability for conformationally dynamic systems. Previously, we demonstrated the use of drift tube ion mobility-mass spectrometry (DT IM-MS) and electron capture dissociation (ECD) to study the metamorphic protein lymphotactin (Ltn). Ltn exists in equilibrium between distinct monomeric (Ltn10) and dimeric (Ltn40) folds, both of which can be preserved and probed in the gas-phase. Here, we further test this mass spectrometric framework, by examining two site directed mutants of Ltn, designed to stabilise either distinct fold in solution, in addition to a truncated form consisting of a minimum model of structure for Ltn10. The truncated mutant has similar collision cross sections to the wild type (WT), for low charge states, and is resistant to ECD fragmentation. The monomer mutant (CC3) presents in similar conformational families as observed previously for the WT Ltn monomer. As with the WT, the CC3 mutant is resistant to ECD fragmentation at low charge states. The dimer mutant W55D is found here to exist as both a monomer and dimer. As a monomer W55D exhibits similar behaviour to the WT, but as a dimer presents a much larger charge state and collision cross section range than the WT dimer, suggesting a smaller interaction interface. In addition, ECD on the W55D mutant yields greater fragmentation than for the WT, suggesting a less stable beta-sheet core. The results highlight the power of MS to provide insight into dynamic proteins, providing further information on each distinct fold of Ltn. In addition we observe differences in the fold stability following single or double point mutations. This approach, therefore, has potential to be a useful tool to screen for the structural effects of mutagenesis, even when sample is limited.

The direct determination of the overall topology and inter-subunit contacts of protein complexes plays an integral role in understanding how different subunits assemble into biologically relevant multisubunit complexes. Mass spectrometry has emerged as a useful structural biological tool because of its sensitivity, high tolerance for heterogeneous mixtures and the fact that crystals are not required. Perturbation of subunit interfaces in solution followed by gas-phase detection using mass spectrometry is a current means of probing the disassembly and hence assembly of protein complexes. Herein, we present an alternative method that employs native mass spectrometry coupled with ion mobility and two stages of surface induced dissociation (SID) where protein complexes are dissociated into subcomplexes in the first SID stage. The subcomplexes are then separated by ion mobility and subsequently fragmented into their individual monomers in the second SID stage (SID-IM-SID), providing information on how individual subunits assemble into protein complexes with different native topologies. The results also illustrate complex dependent differences in charge redistribution onto individual monomers obtained in SID-IM-SID.

A mass spectrometer provides an ideal laboratory to probe the structure and stability of isolated protein ions. Interrogation of each discrete mass/charge-separated species enables the determination of the intrinsic stability of a protein fold, gaining snapshots of unfolding pathways. In solution, the metamorphic protein lymphotactin (Ltn) exists in equilibrium between two distinct conformations, a monomeric (Ltn10) and a dimeric (Ltn40) fold. Here, we use electron capture dissociation (ECD) and drift tube ion mobility-mass spectrometry (DT IM-MS) to analyze both forms and use molecular dynamics (MD) to consider how the solution fold alters in a solvent-free environment. DT IM-MS reveals significant conformational flexibility for the monomer, while the dimer appears more conformationally restricted. These findings are supported by MD calculations, which reveal how salt bridges stabilize the conformers in vacuo. Following ECD experiments, a distinctive fragmentation pattern is obtained for both the monomer and dimer. Monomer fragmentation becomes more pronounced with increasing charge state especially in the disordered regions and C-terminal α-helix in the solution fold. Lower levels of fragmentation are seen in the β-sheet regions and in regions that contain salt bridges, identified by MD simulations. The lowest charge state of the dimer for which we obtain ECD data ([D+9H](9+)) exhibits extensive fragmentation with no relationship to the solution fold and has a smaller collision cross section (CCS) than charge states 10-13+, suggesting a "collapsed" encounter complex. Other charge states of the dimer, as for the monomer, are resistant to fragmentation in regions of β-sheets in the solution fold. This study provides evidence for preservation and loss of global fold and secondary structural elements, providing a tantalizing glimpse into the power of the emerging field of native top-down mass spectrometry.

The use of ion mobility mass spectrometry has grown rapidly over the last two decades. This powerful analytical platform now forms an attractive prospect for comprehensive analysis of many different molecular species, including chemically complex biological molecules. This paper describes the application of IM-MS to the study of peptides. We focus on three different ion mobility devices that are most frequently found in tandem with mass spectrometers. These are instruments using linear drift tubes (LDT), those using travelling wave ion guides (TWIGS) and those employing high field asymmetric ion mobility spectrometry (FAIMS). Each technique is described. Examples are given on the use of IM-MS for the determination of peptide structure, the study of peptides that form amyloid fibrils, and the study of complex peptide mixtures in proteomic investigations. We describe and comment on the methodologies used and the outlook for this developing analytical technique.

Prediction of tandem mass spectrometric (MS/MS) fragmentation for non-peptidic molecules based on structure is of immense interest to the mass spectrometrist. If a reliable approach to MS/MS prediction could be achieved its impact within the pharmaceutical industry could be immense. Many publications have stressed that the fragmentation of a molecular ion or protonated molecule is a complex process that depends on many parameters, making prediction difficult. Commercial prediction software relies on a collection of general heuristic rules of fragmentation, which involve cleaving every bond in the structure to produce a list of 'expected' masses which can be compared with the experimental data. These approaches do not take into account the thermodynamic or molecular orbital effects that impact on the molecule at the point of protonation which could influence the potential sites of bond cleavage based on the structural motif. A series of compounds have been studied by examining the experimentally derived high-resolution MS/MS data and comparing it with the in silico modelling of the neutral and protonated structures. The effect that protonation at specific sites can have on the bond lengths has also been determined. We have calculated the thermodynamically most stable protonated species and have observed how that information can help predict the cleavage site for that ion. The data have shown that this use of in silico techniques could be a possible way to predict MS/MS spectra.

Mass spectrometry has emerged as a useful tool in the study of proteins and protein complexes. It is of fundamental interest to explore how the structures of proteins and protein complexes are affected by the absence of solvent and how this alters with increasing time in the gas phase. Here we demonstrate that a range of protein and protein complexes can be confined within the Trap T-wave region of a modified Waters Synapt G2S instrument, including monomeric (β-lactoglobulin), dimeric (β-lactoglobulin and enolase), tetrameric (streptavidin, concanavalin A, and pyruvate kinase), and pentameric (C-reactive protein) complexes, ranging in size up to 237 kDa. We demonstrate that complexes can be confined within the Trap region for varying lengths of time over the range 1-60 s and with up to 86% trapping efficiency for 1 s trapping. Furthermore, using model systems, we show that these noncovalent complexes can also be fragmented by surface-induced dissociation (SID) following trapping. SID reveals similar dissociation patterns over all trapping times studied for unactivated protein complexes, suggesting that any conformational changes occurring over this time scale are insufficient to cause substantial differences in the SID spectra of these complexes. Intentional alteration of structure by cone activation produces a distinct SID spectrum, with the differences observed being conserved, in comparison to unactivated complex, after trapping. However, subtle differences in the SID spectra of the activated complex are also observed as a function of trapping time.

Transmission electron microscopy, mass spectrometry, and drift tube ion mobility-mass spectrometry are used to study the assemblies formed by the metamorphic chemokine lymphotactin in the presence of a model pentameric glycosaminoglycan, fondaparinux. This combination of techniques delineates significant differences in the complexes observed for two forms of the full length protein as well as a truncated form, without the intrinsically disordered C-terminal tail, over a length scale from few nm to μm assemblies.

In their natural environment, membrane proteins are surrounded by lipids, but the effect that the lipids have on the proteins is not easy to assess. Now, controlling the extent of delipidation has enabled the study of these interactions.

Fragmentation of molecules under collision-induced dissociation (CID) conditions is not well-understood. This may make interpretation of MSMS spectra difficult and limit the effectiveness of software tools intended to aid mass spectral interpretation. Density Functional Theory (DFT) has been successfully applied to explain the thermodynamics of fragmentation in the gas phase by the modelling the effect that protonation has on the bond lengths (and hence bond strengths). In this study, dofetilide and four methylated analogues were used to investigate further the potential for using DFT to understand and predict the CID fragmentation routes. The products ions present in the CID spectra of all five compounds were consistent with charge-directed fragmentation, with protonation adjacent to the cleavage site being required to initiate fragmentation. Protonation at the dissociative site may have occurred either directly or via proton migration. A correlation was observed between protonation-induced bond lengthening and the bonds which were observed to break in the CID spectra. This correlation was quantitative in that the bonds calculated to elongate to the greatest extent gave rise to the most abundant of the major product ions. Thus such quantum calculations may offer the potential for contributing to a predictive tool for aiding the accuracy and speed mass spectral interpretation by generating numerical data in the form of bond length increases to act as descriptors flagging potential bond cleavages.

The leucine zipper interaction between MAX and c-MYC has been studied using mass spectrometry and drift time ion mobility mass spectrometry (DT IM-MS) in addition to circular dichroism spectroscopy. Peptides comprising the leucine zipper sequence with (c-MYC-Zip residues 402-434) and without a postulated small-molecule binding region (c-MYC-ZipΔDT residues 406-434) have been synthesized, along with the corresponding MAX leucine zipper (MAX-Zip residues 74-102). c-MYC-Zip:MAX-Zip complexes are observed both in the absence and in the presence of the reported small-molecule inhibitor 10058-F4 for both forms of c-MYC-Zip. DT IM-MS, in combination with molecular dynamics (MD), shows that the c-MYC-Zip:MAX-Zip complex [M+5H](5+) exists in two conformations, one extended with a collision cross section (CCS) of 1164 ± 9.3 A(2) and one compact with a CCS of 982 ± 6.6 A(2); similar values are observed for the two forms of c-MYC-ZipΔDT:MAX-Zip. Candidate geometries for the complexes have been evaluated with MD simulations. The helical leucine zipper structure previously determined from NMR measurements (Lavigne, P.; et al. J. Mol. Biol. 1998, 281, 165), altered to include the DT region and subjected to a gas-phase minimization, yields a CCS of 1247 A(2), which agrees with the extended conformation we observe experimentally. More extensive MD simulations provide compact complexes which are found to be highly disordered, with CCSs that correspond to the compact form from experiment. In the presence of the ligand, the leucine zipper conformation is completely inhibited and only the more disordered species is observed, providing a novel method to study the effect of interactions of disordered systems and subsequent inhibition of the formation of an ordered helical complex.

Silica sorbents, based on mesoporous crystalline material-41 (MCM-41), were functionalised using mercaptopropyl (MP) or diethylenetriamine (DETA) to extract mercury (II) ions from water. MP-MCM-41 is an extremely efficient and selective sorbent for the removal of mercury (II) from samples of distilled water doped with heavy metal ions and additionally from more complex matrices including tap and river water. In contrast DETA-MCM-41 preferentially removes hard metal ions (chromium, manganese, lead and zinc) over soft metal ions such as mercury. During extraction, the influence of pH on adsorption capacity was examined; a maximum adsorption capacity of 1245 μmol g(-1) was achieved for MP-MCM-41 even at pH values as low as 3. Significantly, a method has been developed for the first time to remove Hg (II) from loaded MP-MCM-41 allowing this analyte to be selectively recovered from water contaminated with a wide range of heavy metal ions. The regeneration method does not disrupt the chelating agent which remains on the surface of the silica permitting reuse of the sorbent in further extractions.