Hybrid Mass Spectrometry Applied across the Production of Antibody Biotherapeutics.

Post expression from the host cells, biotherapeutics undergo downstream processing steps before final formulation. Mass spectrometry and biophysical characterization methods are valuable for examining conformational and stoichiometric changes at these stages, although typically not used in biomanufacturing, where stability is assessed via bulk property studies. Here we apply hybrid MS methods to understand how solution condition changes impact the structural integrity of a biopharmaceutical across the processing pipeline.

Nanohoops Favour Light-Induced Energy Transfer over Charge Separation in Porphyrin/[10]CPP/Fullerene Rotaxanes.

[2]Rotaxanes offer unique opportunities for studying and modulating charge separation and energy transfer, because the mechanical bond allows the robust, yet spatially dynamic tethering of photoactive groups. In this work, we synthesized [2]rotaxane triads comprising a central (aza)[10]CPP⊃C bis-adduct complex and two zinc porphyrin stoppers to address how the movable nanohoop affects light-induced charge separation and energy transfer between the rotaxane subcomponents. We found that neither the parent nanohoop [10]CPP nor its electron-deficient analogue aza[10]CPP actively participate in charge separation.

How storage post sampling influences the stability of sebum when used for mass spectrometry metabolomics analysis?

Sebum is a biofluid excreted by sebaceous glands in the skin. In recent years sebum has been shown to contain endogenous metabolites diagnostic of disease, with remarkable results for Parkinson's Disease. Given that sebum sampling is facile and non-invasive, its potential for use in clinical biochemistry diagnostic assays should be explored including the parameters for standard operating procedures around collection, transport, and storage.

Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins.

Within the intricate landscape of the proteome, approximately 30% of all proteins bind metal ions. This repertoire is even larger when considering all the different forms of a protein, known as proteoforms. Here, we propose the term "metalloforms" to refer to different structural or functional variations of a protein resulting from the binding of various hetero- or homogeneous metal ions.

Conformational Landscapes and Energetics of Carbon Nanohoops and their Ring-in-Ring Complexes.

Carbon nanohoops are promising precursors for the synthesis of nanotubes, whose structural dynamics are not well understood. Here, we investigate the conformational landscape and energetics of cycloparaphenylenes (CPPs), a methylene-bridged CPP and a carbon nanobelt. These nanohoops can form host-guest complexes with other rings, and understanding their structure is crucial for predicting their properties and identifying potential applications.

Exploring the Conformational Landscape of Poly(l-lysine) Dendrimers Using Ion Mobility Mass Spectrometry.

Ion mobility mass spectrometry (IM-MS) measures the mass, size, and shape of ions in the same experiment, and structural information is provided via collision cross-section (CCS) values. The majority of commercially available IM-MS instrumentation relies on the use of CCS calibrants, and here, we present data from a family of poly(l-lysine) dendrimers and explore their suitability for this purpose. In order to test these compounds, we employed three different IM-MS platforms (Agilent 6560 IM-QToF, Waters Synapt G2, and a home-built variable temperature drift tube IM-MS) and used them to investigate six different generations of dendrimers in two buffer gases (helium and nitrogen).

Studying Cation Exchange in {CrCo} Pseudorotaxanes: Preparatory Studies for Making Hybrid Molecular Machines.

In the design of dynamic supramolecular systems used in molecular machines, it is important to understand the binding preferences between the macrocycle and stations along the thread. Here, we apply H NMR spectroscopy to investigate the relative stabilities of a series of linear alkylammonium templated pseudorotaxanes with the general formula [HNRR'][CrCoF(OCCH Bu)] by exchanging the cation in solution. Our results show that the pseudorotaxanes are able to exchange threads via a dissociative mechanism.

Ion Mobility Mass Spectrometry for Large Synthetic Molecules: Expanding the Analytical Toolbox.

Understanding the composition, structure and stability of larger synthetic molecules is crucial for their design, yet currently the analytical tools commonly used do not always provide this information. In this perspective, we show how ion mobility mass spectrometry (IM-MS), in combination with tandem mass spectrometry, complementary techniques and computational methods, can be used to structurally characterize synthetic molecules, make and predict new complexes, monitor disassembly processes and determine stability. Using IM-MS, we present an experimental and computational framework for the analysis and design of complex molecular architectures such as (metallo)supramolecular cages, nanoclusters, interlocked molecules, rotaxanes, dendrimers, polymers and host-guest complexes.

Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant -Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an -Alkylguanine Adductome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, -methylguanine -methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA -alkylguanines (-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple -alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different -alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known -MethylG (-MeG) levels.

Hexanuclear Ln L 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.

Bile salts enhance the susceptibility of the peach allergenic lipid transfer protein, Pru p 3, to in vitro gastrointestinal proteolysis.

Sensitisation to the lipid transfer protein Pru p 3 is associated with severe allergic reactions to peach, the proteins stability being thought to play a role in its allergenicity. Lipid binding increases susceptibility of Pru p 3 to digestion and so the impact of bile salts on the in vitro gastrointestinal digestibility of Pru p 3 was investigated and digestion products mapped by SDS-PAGE and mass spectrometry. Bile salts enhanced the digestibility of Pru p 3 resulting in an ensemble of around 100 peptides spanning the protein's sequence which were linked by disulphide bonds into structures of ~ 5-6 kDa.

Structural Characterization of Cu(I)/Zn(II)-metallothionein-3 by Ion Mobility Mass Spectrometry and Top-Down Mass Spectrometry.

Mammalian zinc metallothionein-3 (ZnMT3) plays an important role in protecting against copper toxicity by scavenging free Cu(II) ions or removing Cu(II) bound to β-amyloid and α-synuclein. While previous studies reported that ZnMT3 reacts with Cu(II) ions to form Cu(I)Zn(II)MT3ox containing two disulfides (ox), the precise localization of the metal ions and disulfides remained unclear. Here, we undertook comprehensive structural characterization of the metal-protein complexes formed by the reaction between ZnMT3 and Cu(II) ions using native ion mobility mass spectrometry (IM-MS).

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.

No skin off your back: the sampling and extraction of sebum for metabolomics.

Introduction: Sebum-based metabolomics (a subset of "sebomics") is a developing field that involves the sampling, identification, and quantification of metabolites found in human sebum. Sebum is a lipid-rich oily substance secreted by the sebaceous glands onto the skin surface for skin homeostasis, lubrication, thermoregulation, and environmental protection. Interest in sebomics has grown over the last decade due to its potential for rapid analysis following non-invasive sampling for a range of clinical and environmental applications.

Ion mobility mass spectrometry and molecular dynamics simulations unravel the conformational stability of zinc metallothionein-2 species.

Ion mobility-mass spectrometry (IM-MS) unraveled different conformational stability in Zn-metallothionein-2. We introduced a new molecular dynamics simulation approach that permitted the exploration of all of the conformational space confirming the experimental data, and revealed that not only the Zn-S bonds but also the α-β domain interactions modulate protein unfolding.

Ion Mobility Mass Spectrometry (IM-MS) for Structural Biology: Insights Gained by Measuring Mass, Charge, and Collision Cross Section.

The investigation of macromolecular biomolecules with ion mobility mass spectrometry (IM-MS) techniques has provided substantial insights into the field of structural biology over the past two decades. An IM-MS workflow applied to a given target analyte provides mass, charge, and conformation, and all three of these can be used to discern structural information. While mass and charge are determined in mass spectrometry (MS), it is the addition of ion mobility that enables the separation of isomeric and isobaric ions and the direct elucidation of conformation, which has reaped huge benefits for structural biology.

Adduct Ions as Diagnostic Probes of Metallosupramolecular Complexes Using Ion Mobility Mass Spectrometry.

Following electrospray ionization, it is common for analytes to enter the gas phase accompanied by a charge-carrying ion, and in most cases, this addition is required to enable detection in the mass spectrometer. These small charge carriers may not be influential in solution but can markedly tune the analyte properties in the gas phase. Therefore, measuring their relative influence on the target molecule can assist our understanding of the structure and stability of the analyte.

Disassembly Mechanisms and Energetics of Polymetallic Rings and Rotaxanes.

Understanding the fundamental reactivity of polymetallic complexes is challenging due to the complexity of their structures with many possible bond breaking and forming processes. Here, we apply ion mobility mass spectrometry coupled with density functional theory to investigate the disassembly mechanisms and energetics of a family of heterometallic rings and rotaxanes with the general formula [NHRR'][CrMF(OCBu)] with M = Mn, Fe, Co, Ni, Cu, Zn, Cd. Our results show that their stability can be tuned both by altering the d-metal composition in the macrocycle and by the end groups of the secondary ammonium cation [NHRR'].

Paper Spray Ionization Ion Mobility Mass Spectrometry of Sebum Classifies Biomarker Classes for the Diagnosis of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, and identification of robust biomarkers to complement clinical diagnosis will accelerate treatment options. Here, we demonstrate the use of direct infusion of sebum from skin swabs using paper spray ionization coupled with ion mobility mass spectrometry (PS-IM-MS) to determine the regulation of molecular classes of lipids in sebum that are diagnostic of PD. A PS-IM-MS method for sebum samples that takes 3 min per swab was developed and optimized.

Untargeted saliva metabolomics by liquid chromatography-Mass spectrometry reveals markers of COVID-19 severity.

Background: The COVID-19 pandemic is likely to represent an ongoing global health issue given the potential for new variants, vaccine escape and the low likelihood of eliminating all reservoirs of the disease. Whilst diagnostic testing has progressed at a fast pace, the metabolic drivers of outcomes-and whether markers can be found in different biofluids-are not well understood. Recent research has shown that serum metabolomics has potential for prognosis of disease progression.

Metabolomics Markers of COVID-19 Are Dependent on Collection Wave.

The effect of COVID-19 infection on the human metabolome has been widely reported, but to date all such studies have focused on a single wave of infection. COVID-19 has generated numerous waves of disease with different clinical presentations, and therefore it is pertinent to explore whether metabolic disturbance changes accordingly, to gain a better understanding of its impact on host metabolism and enable better treatments. This work used a targeted metabolomics platform (Biocrates Life Sciences) to analyze the serum of 164 hospitalized patients, 123 with confirmed positive COVID-19 RT-PCR tests and 41 providing negative tests, across two waves of infection.

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.

Baseline proteomics characterisation of the emerging host biomanufacturing organism Halomonas bluephagenesis.

Despite its greener credentials, biomanufacturing remains financially uncompetitive compared with the higher carbon emitting, hydrocarbon-based chemical industry. Replacing traditional chassis such as E. coli with novel robust organisms, are a route to cost reduction for biomanufacturing.