Sixty years of research on bracken fern (Pteridium spp.) toxins: Environmental exposure, health risks and recommendations for bracken fern control.

Bracken fern (Pteridium spp.) is a highly problematic plant worldwide due to its toxicity in combination with invasive properties on former farmland, in deforested areas and on disturbed natural habitats. The carcinogenic potential of bracken ferns has caused scientific and public concern for six decades. Its genotoxic effects are linked to illudane-type glycosides (ITGs), their aglycons and derivatives. Ptaquiloside is considered the dominating ITG, but with significant contributions from other ITGs. The present review aims to compile evidence regarding environmental pollution by bracken fern ITGs, in the context of their human and animal health implications. The ITG content in bracken fern exhibits substantial spatial, temporal, and chemotaxonomic variation. Consumption of bracken fern as food is linked to human gastric cancer but also causes urinary bladder cancers in bovines browsing on bracken. Genotoxic metabolites are found in milk and meat from bracken fed animals. ITG exposure may also take place via contaminated water with recent data pointing to concentrations at microgram/L-level following rain events. Airborne ITG-exposure from spores and dust has also been documented. ITGs may synergize with major biological and environmental carcinogens like papillomaviruses and Helicobacter pylori to induce cancer, revealing novel instances of chemical and biological co-carcinogenesis. Thus, the emerging landscape from six decades of bracken research points towards a global environmental problem with increasingly complex health implications.

Enhancing Biomolecule Analysis and 2DMS Experiments by Implementation of (Activated Ion) 193 nm UVPD on a FT-ICR Mass Spectrometer.

Ultraviolet photodissociation is a fast, photon-mediated fragmentation method that yields high sequence coverage and informative cleavages of biomolecules. In this work, 193 nm UVPD was coupled with a 12 Tesla FT-ICR mass spectrometer and 10.6 µm infrared multi-photon dissociation to provide gentle slow-heating of UV-irradiated ions. No internal instrument hardware modifications were required. Adjusting the timing of laser pulses to the ion motion within the ICR cell provided consistent fragmentation yield shot-to-shot and may also be used to monitor ion positions within the ICR cell. Single-pulse UVPD of the native-like 5+ charge state of ubiquitin resulted in 86.6% cleavage coverage. Additionally, IR activation post UVPD doubled the overall fragmentation yield and boosted the intensity of UVPD-specific x-type fragments up to 4-fold. This increased yield effect was also observed for the 6+ charge state of ubiquitin, albeit less pronounced. This indicates that gentle slow-heating serves to sever tethered fragments originating from non-covalently linked compact structures and makes activation post UVPD an attractive option to boost fragmentation efficiency for top-down studies. Lastly, UVPD was implemented and optimized as a fragmentation method for 2DMS, a data-independent acquisition method. UVPD-2DMS was demonstrated to be a viable method using BSA digest peptides as a model system.

Fine Structure in Isotopic Peak Distributions Measured Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: A Comparison between an Infinity ICR Cell and a Dynamically Harmonized ICR Cell.

The fine structure of isotopic peak distributions of glutathione in mass spectra is measured using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) at 12 and 15 T magnetic field, with an infinity cell and a dynamically harmonized cell (DHC) respectively. The resolved peaks in the fine structure of glutathione consist of 2H, 13C, 15N, 17O, 18O, 33S, 34S, 36S, and combinations of them. The positions of the measured fine structure peaks agree with the simulated isotopic distributions with the mass error less than 250 ppb in broadband mode for the infinity cell and no more than 125 ppb with the DHC after internal calibration. The 15 T FT-ICR MS with DHC cell also resolved around 30 isotopic peaks in broadband with a resolving power (RP) of 2 M. In narrowband (m/z 307-313), our current highest RP of 13.9 M in magnitude mode was observed with a 36 s transient length by the 15 T FT-ICR MS with the DHC and 2ω detection on the 15 T offers slightly higher RP (14.8 M) in only 18 s. For the 12 T FT-ICR MS with the infinity cell, the highest RP achieved was 15.6 M in magnitude mode with a transient length of 45 s. Peak decay was observed for low abundance peaks, which could be due to the suppression effects from the most abundant peak, as result of ion cloud Coulombic interactions (space-charge).

Revealing the Reactivity of Individual Chemical Entities in Complex Mixtures: the Chemistry Behind Bio-Oil Upgrading.

Bio-oils are precursors for biofuels but are highly corrosive necessitating further upgrading. Furthermore, bio-oil samples are highly complex and represent a broad range of chemistries. They are complex mixtures not simply because of the large number of poly-oxygenated compounds but because each composition can comprise many isomers with multiple functional groups. The use of hyphenated ultrahigh-resolution mass spectrometry affords the ability to separate isomeric species of complex mixtures. Here, we present for the first time, the use of this powerful analytical technique combined with chemical reactivity to gain greater insights into the reactivity of the individual isomeric species of bio-oils. A pyrolysis bio-oils and its esterified bio-oil were analyzed using gas chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry, and in-house software (KairosMS) was used for fast comparison of the hyphenated data sets. The data revealed a total of 10,368 isomers in the pyrolysis bio-oil and an increase to 18,827 isomers after esterification conditions. Furthermore, the comparison of the isomeric distribution before and after esterification provide new light on the reactivities within these complex mixtures; these reactivities would be expected to correspond with carboxylic acid, aldehyde, and ketone functional groups. Using this approach, it was possible to reveal the increased chemical complexity of bio-oils after upgrading and target detection of valuable compounds within the bio-oils. The combination of chemical reactions alongside with in-depth molecular characterization opens a new window for the understanding of the chemistry and reactivity of complex mixtures.

Facile protein conjugation of platinum for light-activated cytotoxic payload release.

The novel Pt(iv) complex trans,trans-[Pt(N3)2(Py)2(OH)(OCO-(PEG)2-NHCSNH-Ph-NCS)] (Pt4) conjugates to the side chain of lysine amino acids in proteins under mild conditions. Reaction with myoglobin generated a bioconjugate that was stable in the dark, but released a Pt(iv) prodrug upon visible light irradiation. A similar procedure was used to conjugate Pt4 to the antibody trastuzumab, resulting in the first photoactivatable Pt(iv)-antibody conjugate, demonstrating potential for highly selective cancer phototherapy.

Solution Condition-Dependent Formation of Gas-Phase Protomers of Alpha-Synuclein in Electrospray Ionization.

One of the main characteristics of biomolecular ions in mass spectrometry is their net charge, and a range of approaches exist to either increase or decrease this quantity in the gas phase. In the context of small molecules, it is well known that, in addition to the charge state, the charge site also has a profound effect on an ion's gas-phase behavior; however, this effect has been far less explored for peptides and intact proteins. Methods exist to determine charge sites of protein ions, and others have observed that the interplay of electrostatic repulsion and inherent basicity leads to different sites gaining or losing a charge depending on the total net charge. Here, we report two distinct protonation site isomers ("protomers") of α-synuclein occurring at the same charge state. The protomers showed important differences in their gas-phase fragmentation behavior and were furthermore distinguishable by ion mobility spectrometry. One protomer was produced under standard electrospray conditions, while the other was observed after addition of 10% dimethyl sulfoxide to the protein solution. Charge sites for both protomers were determined using ultraviolet photodissociation.

Does deamidation affect inhibitory mechanisms towards amyloid protein aggregation?

Deamidated amyloid proteins have been shown to accelerate fibril formation. Herein, the results show the inhibition performance and the interaction site between site-specific inhibitor and amyloid protein are significantly influenced by deamidation; while the inhibition mechanism of non-site specific inhibitor shows no significant disruption caused by amyloid protein deamidation.

Iron stored in ferritin is chemically reduced in the presence of aggregating Aß(1-42).

Atypical low-oxidation-state iron phases in Alzheimer's disease (AD) pathology are implicated in disease pathogenesis, as they may promote elevated redox activity and convey toxicity. However, the origin of low-oxidation-state iron and the pathways responsible for its formation and evolution remain unresolved. Here we investigate the interaction of the AD peptide ß-amyloid (Aß) with the iron storage protein ferritin, to establish whether interactions between these two species are a potential source of low-oxidation-state iron in AD. Using X-ray spectromicroscopy and electron microscopy we found that the co-aggregation of Aß and ferritin resulted in the conversion of ferritin's inert ferric core into more reactive low-oxidation-states. Such findings strongly implicate Aß in the altered iron handling and increased oxidative stress observed in AD pathogenesis. These amyloid-associated iron phases have biomarker potential to assist with disease diagnosis and staging, and may act as targets for therapies designed to lower oxidative stress in AD tissue.

Determination of the Aggregate Binding Site of Amyloid Protofibrils Using Electron Capture Dissociation Tandem Mass Spectrometry.

Amyloid fibril formation is a hallmark in a range of human diseases. Analysis of the molecular details of amyloid aggregation, however, is limited by the difficulties in solubilizing, separating, and identifying the aggregated biomolecules. Additional labeling or protein modification is required in many current analytical techniques in order to provide molecular details of amyloid protein aggregation, but these modifications may result in protein structure disruption. Herein, ultrahigh resolution mass spectrometry (MS) with electron capture dissociation tandem MS (ECD MS/MS) has been applied to monitor the formation of early oligomers of human islet amyloid polypeptide (hIAPP), which aggregate rapidly in the pancreas of type II diabetes (T2D) patients. ECD MS/MS results show the aggregation region of the early oligomers is at the Ser-28/Ser-29 residue of a hIAPP unit and at the Asn-35 residue of another hIAPP unit near the C-terminus in the gas phase. These data contribute to the understanding of the binding site between hIAPP units which may help for specific target region therapeutic development in the future. Furthermore, MS has also been applied to quantify the amount of soluble amyloid protein remaining in the incubated solutions, which can be used to estimate the aggregation rate of amyloid protein during incubation (28 days). These data are further correlated with the results obtained using fluorescence spectroscopy and transmission electron microscopy (TEM) to generate a general overview of amyloid protein aggregation. The methods demonstrated in this article not only explore the aggregation site of hIAPP down to an amino acid residue level, but are also applicable to many amyloid protein aggregation studies.

Metallocomplex-Peptide Interactions Studied by Ultrahigh Resolution Mass Spectrometry.

The OsII arene anticancer complex [(η6-bip)Os(en)Cl]+ (Os1-Cl; where bip = biphenyl and en = ethylenediamine) binds strongly to DNA1 and biomolecules. Here we investigate the interaction between Os1-Cl and the model protein, BSA, using ultrahigh resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS). The specific binding location of Os1 on BSA was investigated with the use of collisionally activated dissociation (CAD) and electron capture dissociation (ECD). CAD MS/MS was found to dissociate the osmium complex from the metallo-peptide complex readily producing unmodified fragments and losing location information. ECD MS/MS, however, successfully retains the osmium modification on the peptides upon fragmentation allowing localization of metallocomplex binding. This study reveals that lysine is a possible binding location for Os1-Cl, apart from the expected binding sites at methionine, histidine, and cysteine. Using a nano liquid chromatography (nLC)-FT-ICR ECD MS/MS study, multiple binding locations, including the N-terminus and C-terminus of digested peptides, glutamic acid, and lysine were also revealed. These results show the multitargeting binding ability of the organo-osmium compound and can be used as a standard workflow for more complex systems, e.g., metallocomplex-cell MS analysis, to evaluate their behavior toward commonly encountered biomolecules.

Generation of maghemite nanocrystals from iron-sulfur centres.

Iron oxide nano-crystals 0.1-1.1 µm in diameter were generated on sulfur-doped amorphous carbon surfaces by electron beam irradiation of the novel 13e- high-spin complex [Fe(4-methyl-1,2-benzenedithiolate)2][NHEt3] encapsulated in a triblock copolymer. Possible relevance to iron nano-mineralization from Fe-S ferredoxin proteins and iron dysregulation in neurological disorders is discussed.

Structural analysis of peptides modified with organo-iridium complexes, opportunities from multi-mode fragmentation.

The most widely used anticancer drugs are platinum complexes, but complexes of other transition metals also show promise and may widen the spectrum of activity, reduce side-effects, and overcome resistance. The latter include organo-iridium(iii) 'piano-stool' complexes. To understand their mechanism of action, it is important to discover how they bind to biomolecules and how binding is affected by functionalisation of the ligands bound to iridium. We have characterised, by MS and MS/MS techniques, unusual adducts from reactions between 3 novel iridium(iii) anti-cancer complexes each possessing reactive sites both at the metal (coordination by substitution of a labile chlorido ligand) and on the ligand (covalent bond formation involving imine formation by one or two aldehyde functions). Peptide modification by the metal complex had a drastic effect on both Collisonally Activated Dissociation (CAD) and Electron Capture Dissociation (ECD) MS/MS behaviour, tuning requirements, and fragmentation channels. CAD MS/MS was effective only when studying the covalent condensation products. ECD MS/MS, although hindered by electron-quenching at the Iridium complex site, was suitable for studying many of the species observed, locating the modification sites, and often identifying them to within a single amino acid residue.

Does deamidation of islet amyloid polypeptide accelerate amyloid fibril formation?

Mass spectrometry has been applied to determine the deamidation sites and the aggregation region of the deamidated human islet amyloid polypeptide (hIAPP). Mutant hIAPP with iso-aspartic residue mutations at possible deamidation sites showed very different fibril formation behaviour, which correlates with the observed deamidation-induced acceleration of hIAPP aggregation.

Sequence-dependent attack on peptides by photoactivated platinum anticancer complexes.

Octahedral platinum(iv) complexes such as trans,trans,trans-[Pt(N3)2(OH)2(pyridine)2] (1) are stable in the dark, but potently cytotoxic to a range of cancer cells when activated by UVA or visible light, and active in vivo. Photoactivation causes the reduction of the complex and leads to the formation of unusual Pt(ii) lesions on DNA. However, radicals are also generated in the excited state resulting from photoactivation (J. S. Butler, J. A. Woods, N. J. Farrer, M. E. Newton and P. J. Sadler, J. Am. Chem. Soc., 2012, 134, 16508-16511). Here we show that once photoactivated, 1 also can interact with peptides, and therefore proteins are potential targets of this candidate drug. High resolution FT-ICR MS studies show that reactions of 1 activated by visible light with two neuropeptides Substance P, RPKPQQFFGLM-NH2 (SubP) and [Lys]3-Bombesin, pEQKLGNQWAVGHLM-NH2 (K3-Bom) give rise to unexpected products, in the form of both oxidised and platinated peptides. Further MS/MS analysis using electron-capture dissociation (ECD) dissociation pathways (enabling retention of the Pt complex during fragmentation), and EPR experiments using the spin-trap DEPMPO, show that the products generated during the photoactivation of 1 depend on the amino acid composition of the peptide. This work reveals the multi-targeting nature of excited state platinum anticancer complexes. Not only can they target DNA, but also peptides (and proteins) by sequence dependent platination and radical mechanisms.

New activation mechanism for half-sandwich organometallic anticancer complexes.

The Cp x C-H protons in certain organometallic RhIII half-sandwich anticancer complexes [(η5-Cp x )Rh(N,N')Cl]+, where Cp x = Cp*, phenyl or biphenyl-Me4Cp, and N,N' = bipyridine, dimethylbipyridine, or phenanthroline, can undergo rapid sequential deuteration of all 15 Cp* methyl protons in aqueous media at ambient temperature. DFT calculations suggest a mechanism involving abstraction of a Cp* proton by the Rh-hydroxido complex, followed by sequential H/D exchange, with the Cp* rings behaving like dynamic molecular 'twisters'. The calculations reveal the crucial role of pπ orbitals of N,N'-chelated ligands in stabilizing deprotonated Cp x ligands, and also the accessibility of RhI-fulvene intermediates. They also provide insight into why biologically-inactive complexes such as [(Cp*)RhIII(en)Cl]+ and [(Cp*)IrIII(bpy)Cl]+ do not have activated Cp* rings. The thiol tripeptide glutathione (γ-l-Glu-l-Cys-Gly, GSH) and the activated dienophile N-methylmaleimide, (NMM) did not undergo addition reactions with the proposed RhI-fulvene, although they were able to control the extent of Cp* deuteration. We readily trapped and characterized RhI-fulvene intermediates by Diels-Alder [4+2] cyclo-addition reactions with the natural biological dienes isoprene and conjugated (9Z,11E)-linoleic acid in aqueous media, including cell culture medium, the first report of a Diels-Alder reaction of a metal-bound fulvene in aqueous solution. These findings will introduce new concepts into the design of organometallic Cp* anticancer complexes with novel mechanisms of action.

Organoiridium Photosensitizers Induce Specific Oxidative Attack on Proteins within Cancer Cells.

Strongly luminescent iridium(III) complexes, [Ir(C,N)2 (S,S)]+ (1) and [Ir(C,N)2 (O,O)] (2), containing C,N (phenylquinoline), O,O (diketonate), or S,S (dithione) chelating ligands, have been characterized by X-ray crystallography and DFT calculations. Their long phosphorescence lifetimes in living cancer cells give rise to high quantum yields for the generation of 1 O2 , with large 2-photon absorption cross-sections. 2 is nontoxic to cells, but potently cytotoxic to cancer cells upon brief irradiation with low doses of visible light, and potent at sub-micromolar doses towards 3D multicellular tumor spheroids with 2-photon red light. Photoactivation causes oxidative damage to specific histidine residues in the key proteins in aldose reductase and heat-shock protein-70 within living cancer cells. The oxidative stress induced by iridium photosensitizers during photoactivation can increase the levels of enzymes involved in the glycolytic pathway.

Comprehensive chemical comparison of fuel composition and aerosol particles emitted from a ship diesel engine by gas chromatography atmospheric pressure chemical ionisation ultra-high resolution mass spectrometry with improved data processing routines.

The analysis of petrochemical materials and particulate matter originating from combustion sources remains a challenging task for instrumental analytical techniques. A detailed chemical characterisation is essential for addressing health and environmental effects. Sophisticated instrumentation, such as mass spectrometry coupled with chromatographic separation, is capable of a comprehensive characterisation, but needs advanced data processing methods. In this study, we present an improved data processing routine for the mass chromatogram obtained from gas chromatography hyphenated to atmospheric pressure chemical ionisation and ultra high resolution mass spectrometry. The focus of the investigation was the primary combustion aerosol samples, i.e. particulate matter extracts, as well as the corresponding fossil fuels fed to the engine. We demonstrate that utilisation of the entire transient and chromatographic information results in advantages including minimisation of ionisation artefacts and a reliable peak assignment. A comprehensive comparison of the aerosol and the feed fuel was performed by applying intensity weighted average values, compound class distribution and principle component analysis. Certain differences between the aerosol generated with the two feed fuels, diesel fuel and heavy fuel oil, as well as between the aerosol and the feed were revealed. For the aerosol from heavy fuel oil, oxidised species from the CHN and CHS class precursors of the feed were predominant, whereas the CHOx class is predominant in the combustion aerosol from light fuel oil. Furthermore, the complexity of the aerosol increases significantly compared to the feed and incorporating a higher chemical space. Coupling of atmospheric pressure chemical ionisation to gas chromatography was found to be a useful additional approach for characterisation of a combustion aerosol, especially with an automated utilisation of the information from the ultra-high resolution mass spectrometer and the chromatographic separation.

Automatic assignment of metal-containing peptides in proteomic LC-MS and MS/MS data sets.

Transition metal-containing proteins and enzymes are critical for the maintenance of cellular function and metal-based (metallo)drugs are commonly used for the treatment of many diseases, such as cancer. Detection and characterisation of metallodrug targets is crucial for improving drug-design and therapeutic efficacy. Due to the unique isotopic ratios of many metal species, and the complexity of proteomic samples, standard MS data analysis of these species is unsuitable for accurate assignment. Herein a new method for differentiating metal-containing species within complex LCMS data is presented based upon the Smart Numerical Annotation Procedure (SNAP). SNAP-LC accounts for the change in isotopic envelopes for analytes containing non-standard species, such as metals, and will accurately identify, record, and display the particular spectra within extended LCMS runs that contain target species, and produce accurate lists of matched peaks, greatly assisting the identification and assignment of modified species and tailored to the metals of interest. Analysis of metallated species obtained from tryptic digests of common blood proteins after reactions with three candidate metallodrugs is presented as proof-of-concept examples and demonstrates the effectiveness of SNAP-LC for the fast and accurate elucidation of metallodrug targets.

Sleep surgery tool: A medical checklist to review prior to operating.

OBJECTIVE: The objective of this study was to systematically review the international literature for studies providing a preoperative checklist for medical disorders to be evaluated or treated before performing sleep surgery. If no checklist exists, then studies providing recommendations would be used to develop a checklist de novo. STUDY DESIGN: Systematic review combined with expert opinion. METHODS: Four databases, including PubMed/Medline were searched through August 10, 2016. RESULTS: 453 potentially relevant studies were screened, 32 were downloaded for full review. No study included a preoperative checklist. No study provided guidance for specific medical disorders to evaluate or screen for prior to sleep surgery. Therefore, we reviewed articles in adults that provided recommendations such as: (1) labs to review, (2) non-operative disorders to evaluate and treat, and (3) comorbidities to optimize prior to performing sleep surgery. These articles were utilized in conjunction with expert opinion to develop a preoperative checklist for surgical guidance. CONCLUSION: There are several items to review prior to performing sleep surgery on obstructive sleep apnea patients. This systematic review and expert opinion-based checklist provides over twenty items for reviewing prior to performing sleep surgery to reduce the chance of operating prematurely.