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We introduce a technique for the directed transfer of molecules from an adjacent reservoir onto a sample surface inside the vacuum chamber of a ToF-SIMS instrument using gas cluster ion beam (GCIB) sputtering. An example application for in situ matrix-enhanced secondary ion mass spectrometry (ME SIMS) is provided. This protocol has attractive features since most modern SIMS instruments are equipped with a GCIB gun. No solvents are required that would delocalize analytes at the surface, and the transfer of matrix molecules can be interlaced with SIMS depth profiling and 3D imaging sputtering and analysis cycles, which is not possible with conventional ME SIMS strategies. The amount of molecular deposition can be finely tuned, which is important for such a surface sensitive technique as SIMS. To demonstrate the concept, we used 2,5-DHB as a matrix for the enhancement of three drug molecules embedded in a tissue homogenate. By automatic operation of sputter deposition and erosion (cleanup) cycles, depth profiling could be achieved with ME SIMS with good repeatability (<4% RSD). Furthermore, we explored several different matrix compounds, including α-CHCA and aqueous solutions of Brønsted acids (formic acid) and 3-nitrobenzonitrile, a volatile compound known to spontaneously produce ions. The latter two matrix compounds were applied at cryogenic measurement conditions, which extend the range of matrices applicable for ME SIMS. Enhancement ratios range from 2 to 13, depending on the analytes and matrix. The method works in principle, but enhancement ratios for the drug molecules are rather limited at this point. Further study and optimization is needed, and the technique introduced here provides a tool to perform systematic studies of matrix compounds and experimental conditions for their potential for signal enhancement in ME SIMS.
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Secondary ion mass spectrometry (SIMS) is gaining popularity for molecular imaging in the life sciences because it is label-free and allows imaging in two and three dimensions. The recent introduction of the OrbiSIMS has significantly improved the utility for biological imaging through combining subcellular spatial resolution with high-performance Orbitrap mass spectrometry. SIMS instruments operate in high-vacuum, and samples are typically analyzed in a freeze-dried state. Consequently, the molecular and structural information may not be well-preserved. We report a method for molecular imaging of biological materials, preserved in a native state, by using an OrbiSIMS instrument equipped with cryogenic sample handling and a high-pressure freezing protocol compatible with mass spectrometry. The performance is demonstrated by imaging a challenging sample (>90% water) of a mature Pseudomonas aeruginosa biofilm in its native state. The 3D distribution of quorum sensing signaling molecules, nucleobases, and bacterial membrane molecules is revealed with high spatial-resolution and high mass-resolution. We discover that analysis in the frozen-hydrated state yields a 10â¯000-fold increase in signal intensity for polar molecules such as amino acids, which has important implications for SIMS imaging of metabolites and pharmaceuticals.
Assuntos
Biofilmes , Pseudomonas aeruginosa/fisiologia , Espectrometria de Massa de Íon Secundário/métodos , Adenina/química , Congelamento , Imageamento Tridimensional , Microscopia Confocal , Percepção de QuorumRESUMO
There is an increasing need in the pharmaceutical industry to reduce drug failure at late stage and thus reduce the cost of developing a new medicine. Since most drug targets are intracellular, this requires a better understanding of the drug disposition within a cell. Secondary ion mass spectrometry has been identified as a potentially important technique to do this, as it is label-free and allows imaging in 3D with subcellular resolution and recent studies have shown promise for amiodarone. An important analytical parameter is sensitivity, and we measure this in a bovine liver homogenate reference sample for 20 drugs representing important class types relevant to the pharmaceutical industry. We also measure the sensitivity for pure drug and show, for the first time, that the secondary ion mass spectrometry (SIMS) positive ionization efficiency for small molecules is a simple power-law relationship to the log P value. This discovery will be important for advancing the understanding of the SIMS ionization process in small molecules that has, until now, been elusive. This simple relationship is found to hold true for drug doped in the bovine liver homogenate reference sample, except for fluticasone, nicardipine, and sorafenib which suffer from severe matrix suppression. This relationship provides a simple semiempirical method to determine drug sensitivity for positive secondary ions. Furthermore, we show, on chosen models, how the use of different solvents during sample preparation can affect the ionization of analytes.
Assuntos
Fígado/química , Preparações Farmacêuticas/química , Espectrometria de Massa de Íon Secundário , Animais , Bovinos , Indústria Farmacêutica , Íons/química , Estrutura Molecular , Peso MolecularRESUMO
Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Bicamadas Lipídicas/química , Nanotecnologia/métodos , Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Fosfolipídeos/química , Engenharia de Proteínas , Espectrometria de Massa de Íon SecundárioRESUMO
Cell-penetrating peptides are promising reagents for gene and drug delivery. They can efficiently traverse the plasma membrane and deliver various cargo materials ranging from genes to nanoparticles. The functional efficiency of cargo often depends on the completeness of intracellular peptide uptake, which can be measured, but its quantification remains largely inconclusive. Existing approaches rely on the use of radioactive and fluorescent labels or tags which allow colorimetric, fluorescent or spectrometric detection, but lack the ability to detect free peptide. Herein we describe a generic label- and tag-free method to measure the concentration of internalised peptide by matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Quantification is preceded by two-dimensional chromatography and is performed at benign temperatures for the lysates of human dermal fibroblasts transfected with cell penetrating peptides in free form. Isotopically labelled peptides of the same structure are used as internal standards to enable accurate determination of concentration of the recovered free peptide. The method offers a minimalistic approach for intracellular quantification, which can be used as a correlative measure for fluorescence-based imaging methods.
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Peptídeos Penetradores de Células/química , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Citoplasma/metabolismo , Humanos , Nanopartículas/uso terapêuticoRESUMO
A VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study for desorption electrospray ionization mass spectrometry (DESI MS) measurements has been conducted with the involvement of 20 laboratories from 10 countries. Participants were provided with an analytical protocol and two reference samples: a thin layer of Rhodamine B and double-sided adhesive tape, each on separate glass slides. The studies comprised acquisition of positive ion mass spectra in predetermined m/z ranges. No sample preparation was required. Results for Rhodamine B show that very consistent craters may be generated. However, inadequacies of the spray and sample stage designs often lead to variable crater shapes. The average repeatability for Rhodamine B is 50%. Yet, repeatabilities better than 20% can be achieved. Rhodamine B proved to be an excellent reference sample to check the sample erosion crater, the sample stage movement and memory effects. Adhesive tape samples show that their average absolute intensity repeatability is 30% and the relative repeatability is 9%. The constancy of these spectra from relative intensities gives day-to-day average relative repeatabilities of 31%, three times worse than the short-term repeatability. Significant differences in the spectra from different laboratories arise from the different adventitious adducts observed or from contaminants that may cause the higher day-to-day variations. It is thought that this may be overcome by allowing some 20 ppb of sodium to be always present in the solvent, to be the dominating adduct. Repeatabilities better than 5% may be achieved with adequate control.
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In this paper, the hydration of a model protein--hen egg white lysozyme in aqueous solution has been presented. The leading method used was FTIR spectroscopy with an application of a technique of semi-heavy water (HDO) isotope dilution. Analysis of spectra of HDO isotopically diluted in water solution of lysozyme allowed us to isolate HDO spectra affected by lysozyme, and thus to characterise the energetic state of water molecules and their arrangement around protein molecules. The number of water molecules and the shape of the affected HDO spectrum were obtained using a classical and a chemometric method. This shape showed that the HDO spectrum affected by lysozyme may be presented as a superposition of two spectra corresponding to HDO affected by N-methylacetamide and the carboxylate anion (of the formic acid). Moreover, based on the difference in intermolecular distances distribution of water molecules (obtained from spectral data), we demonstrated that the lysozyme molecule causes a decrease in population of weak hydrogen bonds, and concurrently increases the probability of an occurrence of short hydrogen bonds in water affected by lysozyme. This conclusion was also confirmed by the molecular dynamics (MD) simulation.
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Óxido de Deutério/química , Simulação de Dinâmica Molecular , Muramidase/química , Água/química , Ligação de Hidrogênio , Modelos Moleculares , Muramidase/metabolismo , Soluções , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The increasing awareness of the significance of microbial biofilms across different sectors is continuously revealing new areas of opportunity in the development of innovative technologies in translational research, which can address their detrimental effects, as well as exploit their benefits. Due to the extent of sectors affected by microbial biofilms, capturing their real financial impact has been difficult. This perspective highlights this impact globally, based on figures identified in a recent in-depth market analysis commissioned by the UK's National Biofilms Innovation Centre (NBIC). The outputs from this analysis and the workshops organised by NBIC on its research strategic themes have revealed the breath of opportunities for translational research in microbial biofilms. However, there are still many outstanding scientific and technological challenges which must be addressed in order to catalyse these opportunities. This perspective discusses some of these challenges.
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BiofilmesRESUMO
The biological activity of therapeutic proteins is strongly dependent on the stability of their folded state, which can easily be compromised by degradation. Oxidation is one of the most common causes of degradation and is typically associated with impairment of the native protein structure. Methionine residues stand out as particularly susceptible to oxidation by reactive oxygen intermediates even under mild conditions. Consequently, methionine oxidation has profound effects on protein activity up to the point of adverse biological responses. Of immediate importance therefore is finding affordable approaches for rapid detection of methionine oxidation before any substantial structural changes can ensue. Herein we report that vibrational bands at 1,044 and 1,113 cm⻹ in the mid-infrared region can serve as characteristic markers of methionine oxidation in oxidatively stressed protein therapeutics, monoclonal antibodies (IgG1 and its antigen-binding fragment). Such Fourier-transform infrared (FTIR) markers underpin rapid detection assays and hold particular promise for correlation of methionine oxidation with protein structure and function.
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Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Metionina/análogos & derivados , Metionina/química , Estresse Oxidativo , Espectroscopia de Infravermelho com Transformada de Fourier , Biomarcadores/análise , Biomarcadores/química , Dicroísmo Circular/métodos , Fragmentos Fab das Imunoglobulinas/análise , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Fragmentos Fc das Imunoglobulinas/análise , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/análise , Imunoglobulina G/uso terapêutico , Metionina/análise , Oxirredução , Relação Quantitativa Estrutura-Atividade , Espectrometria de FluorescênciaRESUMO
A selection of physicochemical and biological assays were investigated for their utility in detecting changes in preparations of Interferon alpha-2a and Interferon alpha-2b (IFN-alpha 2a, IFN-alpha 2b), which had been subjected to stressed conditions, in order to create models of biopharmaceutical products containing product-related impurities. The stress treatments, which included oxidation of methionine residues and storage at elevated temperatures for different periods of time, were designed to induce various degrees of degradation, aggregation or oxidation of the interferon. Biological activity of the stressed preparations was assessed in three different in vitro cell-based bioassay systems: a late-stage anti-proliferative assay and early-stage assays measuring reporter gene activation or endogenous gene expression by quantitative real time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Relevant physicochemical methods such as SDS-PAGE, reverse phase (RP) chromatography, size-exclusion chromatography (SEC) and dynamic light scattering (DLS), proved their complementarity in detecting structural changes in the stressed preparations which were reflected by reductions in biological activity.
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Bioensaio/métodos , Produtos Biológicos/química , Interferon-alfa/análise , Produtos Biológicos/metabolismo , Proliferação de Células , Cromatografia/métodos , Desenho de Fármacos , Eletroforese em Gel de Poliacrilamida , Humanos , Interferon alfa-2 , Interferon-alfa/metabolismo , Metionina/química , Oxigênio/química , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Manejo de Espécimes , TemperaturaRESUMO
Correlative NanoSIMS and EM imaging of amiodarone-treated macrophages shows the internalisation of the drug at a sub-cellular level and reveals its accumulation within the lysosomes, providing direct evidence for amiodarone-induced phospholipidosis. Chemical fixation using tannic acid effectively seals cellular membranes aiding intracellular retention of diffusible drugs.
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Amiodarona/farmacologia , Antiarrítmicos/farmacologia , Macrófagos/efeitos dos fármacos , Nanotecnologia , Fosfolipídeos/metabolismo , Espectrometria de Massa de Íon Secundário , Amiodarona/química , Antiarrítmicos/química , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Lisossomos/química , Lisossomos/metabolismo , Microscopia EletrônicaRESUMO
In this paper we present a chemometric method of analysis leading to isolation of Fourier transform infrared (FT-IR) spectra of biomacromolecules (HEW lysozyme, ctDNA) affected by osmolytes (trimethylamine-N-oxide and N,N,N-trimethylglycine, respectively) in aqueous solutions. The method is based on the difference spectra method primarily used to characterize the structure of solvent affected by solute. The cyclical usage of factor analysis allows precise information to be obtained on the shape of "affected spectra" of analyzed biomacromolecules. "Affected spectra" of selected biomacromolecules give valuable information on their structure in the presence of the osmolytes in solution, as well as on the level of perturbation in dependence of osmolyte concentration. The method also gives a possibility of insight into the mechanism of interaction in presented types of systems. It can be easily adapted to various chemical and biochemical problems where vibrational or ultraviolet-visible (UV-Vis) spectroscopy is used.
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DNA/isolamento & purificação , Muramidase/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Bovinos , Galinhas , DNA/análise , DNA/química , Glicina/análogos & derivados , Glicina/química , Metilaminas/química , Muramidase/análise , Muramidase/químicaRESUMO
The completion of the human genome project has led to intensified efforts toward comprehensive analysis of proteomes. New possibilities exist for efficient proteomic technologies. However, primary attention is given to the discovery of new predictive biomarker patterns. Understanding proteomes and, in particular, protein-mediated interactions underlying their complexity and diversity, is critical for the development of more reliable and robust diagnostic platforms, which are anticipated to enable personalized medicine. Of immediate relevance in this respect are those approaches that capitalize on the application of nanotechnology, which is seen as a powerful tool for the diagnosis of early-stage diseases. Here we highlight the current state of the field exemplified by recent nano-enabled technologies for biomarker discovery.
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Biomarcadores/análise , Nanopartículas/química , Humanos , Nanotecnologia , Neoplasias/diagnóstico , Doenças Neurodegenerativas/diagnóstico , ProteômicaRESUMO
Intensified efforts to decipher the origin of disease at the molecular level stimulate the emergence of more efficient proteomic technologies. To complement this, attempts are being made to identify new predictive biomarkers for building more reliable biomarker patterns. As biomarker research gathers pace an immediate interest becomes focused on platforms, which although based on mainstream approaches, are more amenable to specialist tasks. Particularly relevant this is for disease-specific biomarkers, which are present at very low concentrations in multicomponent biological fluids and require depletion protocols enabling their separation from high-abundance components. In this report, we describe a new strategy allowing the rapid detection of target protein biomarkers by MALDI-ToF mass spectrometry. The approach relies on selective sequestering of target proteins from complex media by engineered microgels, which select proteins by their size (<30 kDa) and isoelectric points (protein pI <6.5). Subsequently, biomarker-loaded microgels are subjected to direct mass-spectrometric analysis without the need for preceding protein extraction. Exemplified by a natural protein-folding motif, coiled-coil, the monitoring of hierarchical folding-dependent macromolecular systems by the approach is also shown. The described strategy offers a general rationale for versatile platforms for high throughput proteomics and holds promise for proteome fingerprinting of biomolecular interactions.