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The pulmonary epithelial glycocalyx is rich in glycosaminoglycans such as hyaluronan and heparan sulfate. Despite their presence, the importance of these glycosaminoglycans in bacterial lung infections remains elusive. To address this, we intranasally inoculated mice with Streptococcus pneumoniae in the presence or absence of enzymes targeting pulmonary hyaluronan and heparan sulfate, followed by characterization of subsequent disease pathology, pulmonary inflammation, and lung barrier dysfunction. Enzymatic degradation of hyaluronan and heparan sulfate exacerbated pneumonia in mice, as evidenced by increased disease scores and alveolar neutrophil recruitment. However, targeting epithelial hyaluronan in combination with Streptococcus pneumoniae infection further exacerbated systemic disease, indicated by elevated splenic bacterial load and plasma levels of pro-inflammatory cytokines. In contrast, enzymatic cleavage of heparan sulfate resulted in increased bronchoalveolar bacterial burden, lung damage and pulmonary inflammation in mice infected with Streptococcus pneumoniae. Accordingly, heparinase-treated mice also exhibited disrupted lung barrier integrity as evidenced by higher alveolar edema scores and vascular protein leakage into the airways. This finding was corroborated in a human alveolus-on-a-chip platform, confirming that heparinase treatment also disrupts the human lung barrier during Streptococcus pneumoniae infection. Notably, enzymatic pre-treatment with either hyaluronidase or heparinase also rendered human epithelial cells more sensitive to pneumococcal-induced barrier disruption, as determined by transepithelial electrical resistance measurements, consistent with our findings in murine pneumonia. Taken together, these findings demonstrate the importance of intact hyaluronan and heparan sulfate in limiting pneumococci-induced damage, pulmonary inflammation, and epithelial barrier function and integrity. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Native mass spectrometry of membrane proteins relies on non-ionic detergents which protect the protein during transfer from solution into the gas phase. Once in the gas phase, the detergent micelle must be efficiently removed, which is usually achieved by collision-induced dissociation (CID). Recently, infrared multiple photon dissociation (IRMPD) has emerged as an alternative activation method for the analysis of membrane proteins, which has led to a growing interest in detergents that efficiently absorb infrared light. Here we investigate whether the absorption properties of synthetic detergents can be tailored by merging structural motifs of existing detergents into new hybrid detergents. We combine gas-phase infrared ion spectroscopy with density functional theory to investigate and rationalize the absorption properties of three established detergents and two hybrid detergents with fused headgroups. We show that, although the basic intramolecular interactions in the parent and hybrid detergents are similar, the three-dimensional structures differ significantly and so do the infrared spectra. Our results outline a roadmap for guiding the synthesis of tailored detergents with computational chemistry for future mass spectrometry applications.
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Tandem mass spectrometry is routinely used for the structural analysis of organic molecules, but many fragmentation reactions are not well understood. Because several potential structures can correspond to a measured mass, the assignment of product ions is ambiguous using mass spectrometry alone. Here, we combine mass spectrometry with high-resolution gas-phase infrared spectroscopy and computational chemistry tools to identify product ion structures and derive collision-induced fragmentation mechanisms of the chromane derivatives Trolox and Methyltrolox. We find that protonated Trolox and Methyltrolox fragment identically via dehydration and decarbonylation, while deprotonated ions display substantially diverging reactivities. For deprotonated Methyltrolox, we observe unusual radical fragmentation reactions and suggest a [1,2]-Wittig rearrangement involving aryl migration in the gas phase. Overall, the combined experimental and theoretical approach presented here revealed complex proton dynamics and intramolecular rearrangement reactions, which expand our understanding on structure-reactivity relationships of isolated molecules in different protonation states.
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Heparin, a widely used clinical anticoagulant, is generally well-tolerated; however, approximately 1% of patients develop heparin-induced thrombocytopenia (HIT), a serious side effect. While efforts to understand the role of chemokines in HIT development are ongoing, certain aspects remain less studied, such as the stabilization of chemokine oligomers by heparin. Here, we conducted a combined ion mobility-native mass spectrometry study to investigate the stability of chemokine oligomers and their complexes with fondaparinux, a synthetic heparin analog. Collision-induced dissociation and unfolding experiments provided clarity on the specificity and relevance of chemokine oligomers and their fondaparinux complexes with varying stoichiometries, as well as the stabilizing effects of fondaparinux binding.
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Anticoagulantes , Fondaparinux , Polissacarídeos , Fondaparinux/química , Fondaparinux/farmacologia , Polissacarídeos/química , Polissacarídeos/metabolismo , Anticoagulantes/química , Anticoagulantes/farmacologia , Quimiocinas/química , Quimiocinas/metabolismo , Humanos , Heparina/química , Heparina/metabolismo , Ligação Proteica , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodosRESUMO
Magnetic resonance elastography (MRE) and diffusion-weighted imaging (DWI) are complementary imaging techniques that detect disease based on viscoelasticity and water mobility, respectively. However, the relationship between viscoelasticity and water diffusion is still poorly understood, hindering the clinical translation of combined DWI-MRE markers. We used DWI-MRE to study 129 biomaterial samples including native and cross-linked collagen, glycosaminoglycans (GAGs) with different sulfation levels, and decellularized specimens of pancreas and liver, all with different proportions of solid tissue, or solid fractions. We developed a theoretical framework of the relationship between mechanical loss and tissue-water mobility based on two parameters, solid and fluid viscosity. These parameters revealed distinct DWI-MRE property clusters characterizing weak, moderate, and strong water-network interactions. Sparse networks interacting weakly with water, such as collagen or diluted decellularized tissue, resulted in marginal changes in water diffusion over increasing solid viscosity. In contrast, dense networks with larger solid fractions exhibited both free and hindered water diffusion depending on the polarity of the solid components. For example, polar and highly sulfated GAGs as well as native soft tissues hindered water diffusion despite relatively low solid viscosity. Our results suggest that two fundamental properties of tissue networks, solid fraction and network polarity, critically influence solid and fluid viscosity in biological tissues. Since clinical DWI and MRE are sensitive to these viscosity parameters, the framework we present here can be used to detect tissue remodeling and architectural changes in the setting of diagnostic imaging. STATEMENT OF SIGNIFICANCE: The viscoelastic properties of biological tissues provide a wealth of information on the vital state of cells and host matrix. Combined measurement of viscoelasticity and water diffusion by medical imaging is sensitive to tissue microarchitecture. However, the relationship between viscoelasticity and water diffusion is still poorly understood, hindering full exploitation of these properties as a combined clinical biomarker. Therefore, we analyzed the parameter space accessible by diffusion-weighted imaging (DWI) and magnetic resonance elastography (MRE) and developed a theoretical framework for the relationship between water mobility and mechanical parameters in biomaterials. Our theory of solid material properties related to particle motion can be translated to clinical radiology using clinically established MRE and DWI.
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Elasticidade , Água , Viscosidade , Água/química , Difusão , Animais , Técnicas de Imagem por Elasticidade/métodos , Humanos , Imagem de Difusão por Ressonância Magnética/métodos , Colágeno/química , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/química , Fígado/diagnóstico por imagemRESUMO
Protonated ions of fucose-containing oligosaccharides are prone to undergo internal glycan rearrangement which results in chimeric fragments that obfuscate mass-spectrometric analysis. Lack of accessible tools that would facilitate systematic analysis of glycans in the gas phase limits our understanding of this phenomenon. In this work, we use density functional theory modeling to interpret cryogenic IR spectra of Lewis a and blood group type H1 trisaccharides and to establish whether these trisaccharides undergo the rearrangement during gas-phase analysis. Structurally unconstrained search reveals that none of the parent ions constitute a thermodynamic global minimum. In contrast, predicted collision cross sections and anharmonic IR spectra provide a good match to available experimental data which allowed us to conclude that fucose migration does not occur in these antigens. By comparing the predicted structures with those obtained for Lewis x and blood group type H2 epitopes, we demonstrate that the availability of the mobile proton and a large difference in the relative stability of the parent ions and rearrangement products constitute the prerequisites for the rearrangement reaction.
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Antígenos do Grupo Sanguíneo de Lewis , Antígenos do Grupo Sanguíneo de Lewis/química , Epitopos/química , Termodinâmica , Polissacarídeos/química , Teoria da Densidade Funcional , Antígenos de Grupos Sanguíneos/química , Espectrofotometria Infravermelho , Oligossacarídeos/química , Trissacarídeos/químicaRESUMO
Glycosaminoglycans (GAGs) are linear and acidic polysaccharides. They are ubiquitous molecules, which are involved in a wide range of biological processes. Despite being structurally simple at first glance, with a repeating backbone of alternating hexuronic acid and hexosamine dimers, GAGs display a highly complex structure, which predominantly results from their heterogeneous sulfation patterns. The commonly applied method for compositional analysis of all GAGs is "disaccharide analysis." In this process, GAGs are enzymatically depolymerized into disaccharides, derivatized with a fluorescent label, and then analysed through liquid chromatography. The limiting factor in the high throughput analysis of GAG disaccharides is the time-consuming liquid chromatography. To address this limitation, we here utilized trapped ion mobility-mass spectrometry (TIM-MS) for the separation of isomeric GAG disaccharides, which reduces the measurement time from hours to a few minutes. A full set of disaccharides comprises twelve structures, with eight possessing isomers. Most disaccharides cannot be differentiated by TIM-MS in underivatized form. Therefore, we developed chemical modifications to reduce sample complexity and enhance differentiability. Quantification is performed using stable isotope labelled standards, which are easily available due to the nature of the performed modifications.
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Dissacarídeos , Glicosaminoglicanos , Dissacarídeos/química , Dissacarídeos/análise , Glicosaminoglicanos/química , Glicosaminoglicanos/análise , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Isomerismo , Cromatografia Líquida/métodosRESUMO
O-glycosylation is a common post-translational modification that is essential for the defensive properties of mucus barriers. Incomplete and altered O-glycosylation is often linked to severe diseases, such as cancer, cystic fibrosis, and chronic obstructive pulmonary disease. Originating from a nontemplate-driven biosynthesis, mucin-type O-glycan structures are very complex. They are often present as heterogeneous mixtures containing multiple isomers. Therefore, the analysis of complex O-glycan mixtures usually requires hyphenation of orthogonal techniques such as liquid chromatography (LC), ion mobility spectrometry, and mass spectrometry (MS). However, MS-based techniques are mainly qualitative. Moreover, LC separation of O-glycans often lacks reproducibility and requires sophisticated data treatment and analysis. Here we present a mucin-type O-glycomics analysis workflow that utilizes hydrophilic interaction liquid chromatography for separation and fluorescence labeling for detection and quantification. In combination with mass spectrometry, a detailed analysis on the relative abundance of specific mucin-type O-glycan compositions and features, such as fucose, sialic acids, and sulfates, is performed. Furthermore, the average number of monosaccharide units of O-glycans in different samples was determined. To demonstrate universal applicability, the method was tested on mucins from different tissue types and mammals, such as bovine submaxillary mucins, porcine gastric mucins, and human milk mucins. To account for day-to-day retention time shifts in O-glycan separations and increase the comparability between different instruments and laboratories, we included fluorescently labeled dextran ladders in our workflow. In addition, we set up a library of glucose unit values for all identified O-glycans, which can be used to simplify the identification process of glycans in future analyses.
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The dense O-glycosylation of mucins plays an important role in the defensive properties of the mucus hydrogel. Aberrant glycosylation is often correlated with inflammation and pathology such as COPD, cancer, and Crohn's disease. The inherent complexity of glycans and the diversity in the O-core structure constitute fundamental challenges for the analysis of mucin-type O-glycans. Due to coexistence of multiple isomers, multidimensional workflows such as LC-MS are required. To separate the highly polar carbohydrates, porous graphitized carbon is often used as a stationary phase. However, LC-MS workflows are time-consuming and lack reproducibility. Here we present a rapid alternative for separating and identifying O-glycans released from mucins based on trapped ion mobility mass spectrometry. Compared to established LC-MS, the acquisition time is reduced from an hour to two minutes. To test the validity, the developed workflow was applied to sputum samples from cystic fibrosis patients to map O-glycosylation features associated with disease.
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Mucinas , Espectrometria de Massas em Tandem , Humanos , Mucinas/metabolismo , Espectrometria de Massas em Tandem/métodos , Reprodutibilidade dos Testes , Polissacarídeos/química , GlicosilaçãoRESUMO
The notion of (anti)aromaticity is a successful concept in chemistry to explain the structure and stability of polycyclic hydrocarbons. Cyclopentadienyl and fluorenyl cations are among the most studied classical antiaromatic systems. In this work, fluorenyl cations are investigated by high-resolution gas-phase infrared spectroscopy in helium droplets. Bare fluorenyl cations are generated in the gas phase by electrospray ionization. After mass-to-charge selection, ions are captured in ultracold helium nanodroplets and probed by infrared spectroscopy using a widely tunable free-electron laser in the 600-1700 cm-1 range. The highly resolved cryogenic infrared spectra confirm, in combination with DFT computations, that all cations are present in their singlet states.
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Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.
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Matriz Extracelular , Papaína , Ratos , Suínos , Humanos , Animais , Matriz Extracelular/química , Papaína/metabolismo , Matriz Extracelular Descelularizada , Pepsina A/análise , Pepsina A/metabolismo , Pepsina A/farmacologia , Proteômica , Hidrogéis/química , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
In past decades, hydrogen bonds involving organic fluorine have been a highly disputed topic. Obtaining clear evidence for the presence of fluorine-specific interactions is generally difficult because of their weak nature. Today, the existence of hydrogen bonds with organic fluorine is widely accepted and supported by numerous studies. However, strong bonds with short Hâ¯F distances remain scarce and are primarily found in designed model compounds. Using a combination of cryogenic gas-phase infrared spectroscopy and density functional theory, we here analyze a series of conformationally unrestrained fluorinated phenylalanine compounds as protonated species. The results suggest proximal NH+â¯F hydrogen bonds with an exceptionally close Hâ¯F distance (1.79 Å) in protonated ortho-fluorophenylalanine.
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Uremic toxins exert pathophysiological effects on cells and tissues, such as the generation of a pro-calcifying subtype of exosome-like extracellular vesicles (EVs) in vascular cells. Little is known about the effects of the toxins on the surface structure of EVs. Thus, we studied the effects of uremic toxins on the abundance of sulfated glycosaminoglycans (GAGs) in EVs, and the implications for binding of ligands such as very small superparamagnetic iron oxide particles (VSOPs) which could be of relevance for radiological EV-imaging. Vascular cells were treated with the uremic toxins NaH2PO4 and a mixture of urea and indoxyl sulfate. Uremia in rats was induced by adenine feeding. EVs were isolated from culture supernatants and plasma of rats. By proton T1-relaxometry, magnetic particle spectroscopy, and analysis of genes, proteins, and GAG-contents, we analyzed the roles of GAGs in the ligand binding of EVs. By influencing GAG-associated genes in host cells, uremic toxins induced higher GAG contents in EVs, particularly of sulfated chondroitin sulfate and heparan sulfate chains. EVs with high GAG content interacted stronger with VSOPs compared to control ones. This was confirmed by experiments with GAG-depleted EVs from genetically modified CHO cells and with uremic rat-derived EVs. Mechanistically, uremic toxin-induced PI3K/AKT-signaling and expression of the sulfate transporter SLC26A2 in host cells contributed to high GAG contents in EVs. In conclusion, uremic conditions induce enhanced GAG contents in EVs, which entails a stronger interaction with VSOPs. VSOPs might be suitable for radiological imaging of EVs rich in GAGs.
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Exossomos , Vesículas Extracelulares , Toxinas Biológicas , Animais , Ratos , Cricetinae , Toxinas Urêmicas , Cricetulus , Fosfatidilinositol 3-Quinases , Glicosaminoglicanos , Nanopartículas Magnéticas de Óxido de FerroRESUMO
Nanoelectrospray ionization (nano-ESI) is a highly efficient and a widely used technique for the ionization of minute amounts of analyte. Offline nano-ESI sources are convenient for the direct infusion of complex mixtures that suffer from high matrix content and are crucial for the native mass spectrometric analysis of proteins. For Bruker instruments, no such source is readily available. Here we close this gap and present a 3D-printable nano-ESI source for Bruker instruments, which can be assembled by anyone with access to 3D printers. The source can be fitted to any Bruker mass spectrometer with an ionBooster ESI source and only requires minor, reversible changes to the original Bruker hardware. The general utility was demonstrated by recording high-resolution MS spectra of small molecules, intact proteins, as well as complex biological samples in negative and positive ion mode on two different Bruker instruments.
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Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.
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Detergentes , Micelas , Espectrometria de Massas/métodos , Proteínas de Membrana , Receptores Acoplados a Proteínas GRESUMO
Ferrier reactions follow a mechanistic pathway whereby Lewis acid activation of a cyclic enol ether facilitates departure of an allylic leaving group to form a glycosyl Ferrier cation. Attack on the Ferrier cation provides a new acetal linkage concurrent with the transposition of the alkene moiety. The idiosyncratic outcomes of Ferrier reactions of seven-membered ring carbohydrate-based oxepines prompted an investigation of its corresponding septanosyl Ferrier cation. Experiments that characterized the ion, including gas-phase cryogenic IR spectroscopy matched with density functional theory-calculated spectra of candidate cation structures, as well as product analysis from solution-phase Ferrier reactions, are reported here. Results from both approaches revealed an inclination of the seven-membered ring cation to contract to five-membered ring structures. Gas-phase IR spectra matched best to calculated spectra of structures in which five-membered dioxolenium formation opened the oxepine ring. In the solution phase, an attack on the ion by water led to an acyclic enal that cyclized to a C-methylene-aldehydo arabinofuranoside species. Attack by allyl trimethylsilane, on the other hand, was diastereoselective and yielded a C-allyl septanoside.
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Glycosaminoglycans (GAGs) are complex polysaccharides exhibiting a vast structural diversity and fulfilling various functions mediated by thousands of interactions in the extracellular matrix, at the cell surface, and within the cells where they have been detected in the nucleus. It is known that the chemical groups attached to GAGs and GAG conformations comprise "glycocodes" that are not yet fully deciphered. The molecular context also matters for GAG structures and functions, and the influence of the structure and functions of the proteoglycan core proteins on sulfated GAGs and vice versa warrants further investigation. The lack of dedicated bioinformatic tools for mining GAG data sets contributes to a partial characterization of the structural and functional landscape and interactions of GAGs. These pending issues will benefit from the development of new approaches reviewed here, namely (i) the synthesis of GAG oligosaccharides to build large and diverse GAG libraries, (ii) GAG analysis and sequencing by mass spectrometry (e.g., ion mobility-mass spectrometry), gas-phase infrared spectroscopy, recognition tunnelling nanopores, and molecular modeling to identify bioactive GAG sequences, biophysical methods to investigate binding interfaces, and to expand our knowledge and understanding of glycocodes governing GAG molecular recognition, and (iii) artificial intelligence for in-depth investigation of GAGomic data sets and their integration with proteomics.
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In recent years, glycosaminoglycans (GAGs) have emerged into the focus of biochemical and biomedical research due to their importance in a variety of physiological processes. These molecules show great diversity, which makes their analysis highly challenging. A promising tool for identifying the structural motifs and conformation of shorter GAG chains is cryogenic gas-phase infrared (IR) spectroscopy. In this work, the cryogenic gas-phase IR spectra of mass-selected heparan sulfate (HS) di-, tetra-, and hexasaccharide ions were recorded to extract vibrational features that are characteristic to structural motifs. The data were augmented with chondroitin sulfate (CS) disaccharide spectra to assemble a training library for random forest (RF) classifiers. These were used to discriminate between GAG classes (CS or HS) and different sulfate positions (2-O-, 4-O-, 6-O-, and N-sulfation). With optimized data preprocessing and RF modeling, a prediction accuracy of >97% was achieved for HS tetra- and hexasaccharides based on a training set of only 21 spectra. These results exemplify the importance of combining gas-phase cryogenic IR ion spectroscopy with machine learning to improve the future analytical workflow for GAG sequencing and that of other biomolecules, such as metabolites.
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Glicosaminoglicanos , Algoritmo Florestas Aleatórias , Glicosaminoglicanos/química , Sulfatos de Condroitina/química , Heparitina Sulfato , Espectrofotometria InfravermelhoRESUMO
Fatty acids are an abundant class of lipids that are characterised by wide structural variation including isomeric diversity arising from the position and configuration of functional groups. Traditional approaches to fatty acid characterisation have combined chromatography and mass spectrometry for a description of the composition of individual fatty acids while infrared (IR) spectroscopy has provided insights into the functional groups and bond configurations at the bulk level. Here we exploit universal 3-pyridylcarbinol ester derivatization of fatty acids to acquire IR spectra of individual lipids as mass-selected gas-phase ions. Intramolecular interactions between the protonated pyridine moiety and carbon-carbon double bonds present highly sensitive probes for regiochemistry and configuration through promotion of strong and predictable shifts in IR resonances. Gas-phase IR spectra obtained from unsaturated fatty acids are shown to discriminate between isomers and enable the first unambiguous structural assignment of 6Z-octadecenoic acid in human-derived cell lines. Compatibility of 3-pyridylcarbinol ester derivatization with conventional chromatography-mass spectrometry and now gas-phase IR spectroscopy paves the way for comprehensive structure elucidation of fatty acids that is sensitive to regio- and stereochemical variations and with the potential to uncover new pathways in lipid metabolism.
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Fucose is a signaling carbohydrate that is attached at the end of glycan processing. It is involved in a range of processes, such as the selectin-dependent leukocyte adhesion or pathogen-receptor interactions. Mass-spectrometric techniques, which are commonly used to determine the structure of glycans, frequently show fucose-containing chimeric fragments that obfuscate the analysis. The rearrangement leading to these fragments-often referred to as fucose migration-has been known for more than 25â years, but the chemical identity of the rearrangement product remains unclear. In this work, we combine ion-mobility spectrometry, radical-directed dissociation mass spectrometry, cryogenic IR spectroscopy of ions, and density-functional theory calculations to deduce the product of the rearrangement in the model trisaccharides Lewis x and blood group H2. The structural search yields the fucose moiety attached to the galactose with an α(1â6) glycosidic bond as the most likely product.