Use of a charge reducing agent to enable intact mass analysis of cysteine-linked antibody-drug-conjugates by native mass spectrometry

AbstractAntibody-drug-conjugates (ADC) are a growing class of anticancer biopharmaceuticals. Conjugation of cysteine linked ADCs, requires initial reduction of mAb inter-chain disulfide bonds, as the drugs are attached via thiol chemistry. This results in the active mAb moiety being transformed from a covalently linked tetramer to non-covalently linked complexes, which hinders precise determination of drug load with LC–MS. Here, we show how the addition of the charge reducing agent triethylammonium acetate (TEAA) preserves the intact mAb structure, is well suited to the study of cysteine linked conjugates and facilitates easy drug load determination by direct infusion native MS

The use of ion mobility mass spectrometry to assist protein design:A case study on zinc finger fold versus coiled coil interactions

A combination of affinity mass spectrometry, collisional activation and ion mobility analysis coupled with molecular dynamics measurements, are used to probe a zinc finger-coiled coil switching peptide system.</p

Recommendations for reporting ion mobility Mass Spectrometry measurements

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method‐dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc

Ion mobility mass spectrometry uncovers the impact of the patterning of oppositely charged residues on the conformational distributions of intrinsically disordered proteins

The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion mobility-mass spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27Kip1. We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, κ14 and κ56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (κ31) and κ14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, κ56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase molecular dynamics simulations demonstrate that the interplay between chain solvation and intrachain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states

The complexity of selection at the major primate β-defensin locus

BACKGROUND: We have examined the evolution of the genes at the major human β-defensin locus and the orthologous loci in a range of other primates and mouse. For the first time these data allow us to examine selective episodes in the more recent evolutionary history of this locus as well as the ancient past. We have used a combination of maximum likelihood based tests and a maximum parsimony based sliding window approach to give a detailed view of the varying modes of selection operating at this locus. RESULTS: We provide evidence for strong positive selection soon after the duplication of these genes within an ancestral mammalian genome. Consequently variable selective pressures have acted on β-defensin genes in different evolutionary lineages, with episodes both of negative, and more rarely positive selection, during the divergence of primates. Positive selection appears to have been more common in the rodent lineage, accompanying the birth of novel, rodent-specific β-defensin genes. These observations allow a fuller understanding of the evolution of mammalian innate immunity. In both the rodent and primate lineages, sites in the second exon have been subject to positive selection and by implication are important in functional diversity. A small number of sites in the mature human peptides were found to have undergone repeated episodes of selection in different primate lineages. Particular sites were consistently implicated by multiple methods at positions throughout the mature peptides. These sites are clustered at positions predicted to be important for the specificity of the antimicrobial or chemoattractant properties of β-defensins. Surprisingly, sites within the prepropeptide region were also implicated as being subject to significant positive selection, suggesting previously unappreciated functional significance for this region. CONCLUSIONS: Identification of these putatively functional sites has important implications for our understanding of β-defensin function and for novel antibiotic design

Hexanuclear Ln6L6 Complex Formation by using an Unsymmetric Ligand

Multinuclear, self‐assembled lanthanide complexes present clear opportunities as sensors and imaging agents. Despite the widely acknowledged potential of this class of supramolecule, synthetic and characterization challenges continue to limit systematic studies into their self‐assembly restricting the number and variety of lanthanide architectures reported relative to their transition metal counterparts. Here we present the first study evaluating the effect of ligand backbone symmetry on multinuclear lanthanide complex self‐assembly. Replacement of a symmetric ethylene linker with an unsymmetric amide at the centre of a homoditopic ligand governs formation of an unusual Ln6L6 complex with coordinatively unsaturated metal centres. The choice of triflate as a counterion, and the effect of ionic radii are shown to be critical for formation of the Ln6L6 complex. The atypical Ln6L6 architecture is characterized using a combination of mass spectrometry, luminescence, DOSY NMR and EPR spectroscopy measurements. Luminescence experiments support clear differences between comparable Eu6L6 and Eu2L3 complexes, with relatively short luminescent lifetimes and low quantum yields observed for the Eu6L6 structure indicative of non‐radiative decay processes. Synthesis of the Gd6L6analogue allows three distinct Gd···Gd distance measurements to be extracted using homo‐RIDME EPR experiments

Identification and proteomic profiling of exosomes in human cerebrospinal fluid

<p>Abstract</p> <p>Background</p> <p>Exosomes are released from multiple cell types, contain protein and RNA species, and have been exploited as a novel reservoir for disease biomarker discovery. They can transfer information between cells and may cause pathology, for example, a role for exosomes has been proposed in the pathophysiology of Alzheimer's disease. Although studied in several biofluids, exosomes have not been extensively studied in the cerebrospinal fluid (CSF) from humans. The objective of this study was to determine: 1) whether human CSF contains exosomes and 2) the variability in exosomal protein content across individuals.</p> <p>Methods</p> <p>CSF was collected from 5 study participants undergoing thoraco-abdominal aortic aneurysm repair (around 200 - 500 ml per participant) and low-density membrane vesicles were concentrated by ultracentrifugation. The presence of exosomes was determined by western blot for marker proteins, isopycnic centrifugation on a sucrose step gradient and transmission electron microscopy with immuno-labelling. Whole protein profiling was performed using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR).</p> <p>Results</p> <p>Flotillin 1 and tumor susceptibility gene 101 (TSG101), two exosomal marker proteins, were identified in the ultracentrifugation pellet using western blot. These markers localized to a density consistent with exosomes following isopycnic centrifugation. Transmission electron microscopy visualized structures consistent with exosomes in size and appearance that labelled positive for flotillin 1. Therefore, the pellet that resulted from ultracentrifugation of human CSF contained exosomes. FT-ICR profiling of this pellet was performed and 84-161 ions were detected per study participant. Around one third of these ions were only present in a single study participant and one third were detected in all five. With regard to ion quantity, the median coefficient of variation was 81% for ions detected in two or more samples.</p> <p>Conclusions</p> <p>Exosomes were identified in human CSF and their proteome is a potential new reservoir for biomarker discovery in neurological disorders such as Alzheimer's disease. However, techniques used to concentrate exosomes from CSF need refinement to reduce variability. In this study we used relatively large starting volumes of human CSF, future studies will focus on exosome isolation from smaller 'real life' clinical samples; a key challenge in the development of exosomes as translational tools.</p

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

From Europe PMC via Jisc Publications RouterHistory: ppub 2021-05-01, epub 2021-05-05Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Grant(s): BB/L002655/1, BB/L016486/1, BB/M011208/1Organisms 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

Peptide Fragments of a β-Defensin Derivative with Potent Bactericidal Activity

β-Defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine β-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this β-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide