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)-3,4HJihydro-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 metalfree 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 dinners, generating more compact peptide conformations than those from EGCGuntreated 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 structurebased mechanism.
MS analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low ...concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other MS approaches in structural biology. Methods such as H/D exchange, oxidative foot‐printing, chemical cross‐linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative MS approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly advancing area.
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.
In this paper, in order to increase the power conversion efficiency we demonstrated the selective growth of “nanoforest” composed of high density, long branched “treelike” multigeneration ...hierarchical ZnO nanowire photoanodes. The overall light-conversion efficiency of the branched ZnO nanowire DSSCs was almost 5 times higher than the efficiency of DSSCs constructed by upstanding ZnO nanowires. The efficiency increase is due to greatly enhanced surface area for higher dye loading and light harvesting, and also due to reduced charge recombination by providing direct conduction pathways along the crystalline ZnO “nanotree” multi generation branches. We performed a parametric study to determine optimum hierarchical ZnO nanowire photoanodes through the combination of both length-wise growth and branched growth processes. The novel selective hierarchical growth approach represents a low cost, all solution processed hydrothermal method that yields complex hierarchical ZnO nanowire photoanodes by utilizing a simple engineering of seed particles and capping polymer.
Positively a gas: Protein structure determination using mass spectrometry can be assisted by the addition of stabilizing cations to the gas‐phase molecule. Densely charged cations (see scheme, green) ...are found to stabilize protein tertiary structure for accurate mass determination of homo‐ or heterogeneous protein complexes. The cations remain tightly bound to the protein throughout the analysis, allowing the protein to remain in a folded state.
High-accuracy, high-resolution ion mobility measurements enable a vast array of important contemporary applications in biological chemistry. With the recent advent of both new, widely available ...commercial instrumentation and also new calibration datasets tailored for the aforementioned commercial instrumentation, the possibilities for extending such high performance measurements to a diverse set of applications have never been greater. Here, we assess the performance characteristics of a second-generation traveling-wave ion mobility separator, focusing on those figures of merit that lead to making measurements of collision cross-section having both high precision and high accuracy. Through performing a comprehensive survey of instrument parameters and settings, we find instrument conditions for optimized drift time resolution, cross-section resolution, and cross-section accuracy for a range of peptide, protein and multi-protein complex ions. Moreover, the conditions for high accuracy IM results are significantly different from those optimized for separation resolution, indicating that a balance between these two metrics must be attained for traveling wave IM separations of biomolecules. We also assess the effect of ion heating during IM separation on instrument performance.
Folded or not? Ion mobility–mass spectrometry investigation of an activated macromolecular protein complex lends insight into the structures of intermediates formed in the dissociation process. The ...activated ions of human tetrameric transthyretin populate partially folded intermediate states (see picture; folded subunits in blue, partially unfolded subunits in red) prior to dissociation.
Pharmacokinetic analysis of antibody-drug conjugates (ADCs) requires characterization and quantification of both the antibody-conjugated cytotoxic drug molecule (acDrug) as well as the antibody ...vehicle, among other analytes, in order to assess the safety and efficacy of ADCs. Due to the complexity of biological matrices, immunoaffinity capture is widely used for enrichment of the biotherapeutic, followed by enzymatic or chemical release of the drug and LC-MS/MS analysis to provide the concentration of acDrug. This bioanalytical strategy has been used successfully with ADCs, but is limited to ADCs having cleavable linkers. Herein, we developed a sensitive and specific method that involved subjecting the ADC to tryptic digestion, and measured a peptide that included cysteine conjugated to the drug to provide quantification of acDrug. Using this method for a THIOMAB™ antibody-drug conjugate (TDC) conjugated to MMAE via a cleavable linker, valine–citrulline, we compared peptide-linker MMAE data from the new assay format with earlier MMAE data for acDrug. This showed that the new assay format provides robust acDrug as well as total antibody concentration to study in vitro stability of the TDC in multiple matrices and in vivo pharmacokinetic models of TDC in rat and mouse. The data from the two orthogonal modes of acDrug analysis showed good agreement with each other, allowing us to successfully quantify acDrug to study the stability in vitro and the pharmacokinetic parameters in vivo. This new assay strategy allows acDrug quantification for ADCs with non-cleavable linkers where the resulting acDrug analyte is a peptide-linker drug.
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.
Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of ...tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes.
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