Use Of Mass Spectrometric Techniques Research Articles

Proteomic identification of Axc, a novel beta-lactamase with carbapenemase activity in a meropenem-resistant clinical isolate of Achromobacter xylosoxidans

The development of antibiotic resistance during treatment is a threat to patients and their environment. Insight in the mechanisms of resistance development is important for appropriate therapy and infection control. Here, we describe how through the application of mass spectrometry-based proteomics, a novel beta-lactamase Axc was identified as an indicator of acquired carbapenem resistance in a clinical isolate of Achromobacter xylosoxidans. Comparative proteomic analysis of consecutively collected susceptible and resistant isolates from the same patient revealed that high Axc protein levels were only observed in the resistant isolate. Heterologous expression of Axc in Escherichia coli significantly increased the resistance towards carbapenems. Importantly, direct Axc mediated hydrolysis of imipenem was demonstrated using pH shift assays and 1H-NMR, confirming Axc as a legitimate carbapenemase. Whole genome sequencing revealed that the susceptible and resistant isolates were remarkably similar. Together these findings provide a molecular context for the fast development of meropenem resistance in A. xylosoxidans during treatment and demonstrate the use of mass spectrometric techniques in identifying novel resistance determinants.

The Use of Mass Spectrometric Techniques to Differentiate Isobaric and Isomeric Flavonoid Conjugates from Axyris amaranthoides.

Flavonoids are a group of compounds that are commonly found in various plants, where they play important roles in many processes, including free radical scavenging and UV protection. These compounds can also act as chemical messengers, physiological regulators or protectants against pathogens in the defense reactions of plants. Flavonoid activity is regulated by the addition of various substituents, usually mono- or oligosaccharides of common sugars, such as glucose, rhamnose or galactose. In some plants, glucuronic acid is attached, and this sugar is often acylated by phenylpropanoic acids. Identification of these compounds and their derivatives is of great importance to understanding their role in plant metabolism and defense mechanisms; this research is important because flavonoids are frequently a significant constituent of the human diet. In this study, we identify the flavonoid conjugates present in Axyris amaranthoides L. extracts and demonstrate the usefulness of high-resolution mass spectrometry (HRMS) analyzers for the differentiation of isobaric compounds and the utility of fragmentation spectra for the differentiation of isomeric structures. According to our knowledge, some of the structures, especially dehydrodiferulated conjugates of tricin, whose structures are proposed here have been found for the first time in plant material.

The Evolving Contribution of Mass Spectrometry to Integrative Structural Biology.

Protein complexes are key catalysts and regulators for the majority of cellular processes. Unveiling their assembly and structure is essential to understanding their function and mechanism of action. Although conventional structural techniques such as X-ray crystallography and NMR have solved the structure of important protein complexes, they cannot consistently deal with dynamic and heterogeneous assemblies, limiting their applications to small scale experiments. A novel methodological paradigm, integrative structural biology, aims at overcoming such limitations by combining complementary data sources into a comprehensive structural model. Recent applications have shown that a range of mass spectrometry (MS) techniques are able to generate interaction and spatial restraints (cross-linking MS) information on native complexes or to study the stoichiometry and connectivity of entire assemblies (native MS) rapidly, reliably, and from small amounts of substrate. Although these techniques by themselves do not solve structures, they do provide invaluable structural information and are thus ideally suited to contribute to integrative modeling efforts. The group of Brian Chait has made seminal contributions in the use of mass spectrometric techniques to study protein complexes. In this perspective, we honor the contributions of the Chait group and discuss concepts and milestones of integrative structural biology. We also review recent examples of integration of structural MS techniques with an emphasis on cross-linking MS. We then speculate on future MS applications that would unravel the dynamic nature of protein complexes upon diverse cellular states.Graphical ᅟ

Cationicity-enhanced analogues of the antimicrobial peptides, AcrAP1 and AcrAP2, from the venom of the scorpion, Androctonus crassicauda, display potent growth modulation effects on human cancer cell lines.

The non disulphide-bridged peptides (NDBPs) of scorpion venoms are attracting increased interest due to their structural heterogeneity and broad spectrum of biological activities. Here, two novel peptides, named AcrAP1 and AcrAP2, have been identified in the lyophilised venom of the Arabian scorpion, Androctonus crassicauda, through “shotgun” molecular cloning of their biosynthetic precursor-encoding cDNAs. The respective mature peptides, predicted from these cloned cDNAs, were subsequently isolated from the same venom sample using reverse phase HPLC and their identities were confirmed by use of mass spectrometric techniques. Both were found to belong to a family of highly-conserved scorpion venom antimicrobial peptides - a finding confirmed through the biological investigation of synthetic replicates. Analogues of both peptides designed for enhanced cationicity, displayed enhanced potency and spectra of antimicrobial activity but, unlike the native peptides, these also displayed potent growth modulation effects on a range of human cancer cell lines. Thus natural peptide templates from venom peptidomes can provide the basis for rational analogue design to improve both biological potency and spectrum of action. The diversity of such templates from such natural sources undoubtedly provides the pharmaceutical industry with unique lead compounds for drug discovery.

Identification and Functional Characterization of 14-3-3 in TLR2 Signaling

The Interleukin-1/Toll-like receptor signaling pathway is a crucial signaling pathway within the innate immune system and the use of mass spectrometric techniques became valuable to investigate signal transduction pathways. To date only a few reports exist that focus on the mass spectrometric identification of novel signaling intermediates within the TLR signal transduction pathway. Here we used this approach systematically to identify new interaction partners of the TLR signaling pathway and subsequently characterized them functionally. We identified 14-3-3 theta as a new member of the TLR signaling complex. With genetic complementation assays, we demonstrate that 14-3-3 negatively regulates TLR2-dependent NF-κB activity and amplifies the TLR4-dependent activation of the transcription factor. While 14-3-3 has no effect on TLR-induced apoptosis in innate immune cells, it controls the release of the inflammatory, IRF3-dependent cytokines like RANTES and IP-10 after stimulation with LPS. Most strikingly, 14-3-3 controls the production of proinflammatory cytokines like IL-6, IL-8, and TNFα in a different manner. Our results identify 14-3-3 theta as a new and important regulatory protein in the TLR signaling suppressing the MyD88-dependent pathway.

Structural Analysis of Fungal Cerebrosides

Of the ceramide monohexosides (CMHs), gluco- and galactosyl-ceramides are the main neutral glycosphingolipids expressed in fungal cells. Their structural determination is greatly dependent on the use of mass spectrometric techniques, including fast atom bombardment-mass spectrometry, electrospray ionization, and energy collision-induced dissociation mass spectrometry. Nuclear magnetic resonance has also been used successfully. Such a combination of techniques, combined with classical analytical separation, such as high-performance thin layer chromatography and column chromatography, has led to the structural elucidation of a great number of fungal CMHs. The structure of fungal CMH is conserved among fungal species and consists of a glucose or galactose residue attached to a ceramide moiety containing 9-methyl-4,8-sphingadienine with an amidic linkage to hydroxylated fatty acids, most commonly having 16 or 18 carbon atoms and unsaturation between C-3 and C-4. Along with their unique structural characteristics, fungal CMHs have a peculiar subcellular distribution and striking biological properties. Fungal cerebrosides were also characterized as antigenic molecules directly or indirectly involved in cell growth or differentiation in Schizophyllum commune, Cryptococcus neoformans, Pseudallescheria boydii, Candida albicans, Aspergillus nidulans, Aspergillus fumigatus, and Colletotrichum gloeosporioides. Besides classical techniques for cerebroside (CMH) analysis, we now describe new approaches, combining conventional thin layer chromatography and mass spectrometry, as well as emerging technologies for subcellular localization and distribution of glycosphingolipids by secondary ion mass spectrometry and imaging matrix-assisted laser desorption ionization time-of-flight.

Identification of free and bound volatile compounds as typicalness and authenticity markers of non-aromatic grapes and wines through a combined use of mass spectrometric techniques

Molecular approaches by means of a combined use of mass spectrometric techniques can be important in order to open new possibilities in the differentiation and defense of typical products; in this study, a possible approach to the analysis of varietal volatile compounds and some precursors of a non-aromatic grape variety (Falanghina cv., Vitis vinifera L.) was traced through a combined use of techniques based on mass spectrometry (GC/MS, LC/ESI-MS, MALDI-TOF-MS). Dominant terpene compounds (limonene, cis-furanlinalool oxide, geraniol, 4-carene, myrcene, linalool, α-terpineol), terpene-derivatives (bornyl acetate, menthol), terpene glycosides (glucosides, arabinosylglucosides and rhamnosylglucosides of linalool and geraniol), and norisoprenoids (β-damascenone) were identified in grapes and monovarietal wines, overcoming the analytical difficulties deriving from the low concentration of these compounds strictly related to the variety. The potential release of varietal volatile compounds from the grapes was also explored by enzyme hydrolysis.

Characterization of acrolein-induced protein cross-links

Lipid peroxidation products contribute to protein aggregation that occurs during oxidative stress in a number of degenerative disorders. Acrolein (ACR), a highly toxic lipid peroxidation aldehyde, is a strong cross-linking agent of cellular components such as proteins. To understand the mechanisms of oxidative stress-induced protein aggregation, this study characterized the ACR modification of chain B from bovine insulin by mass spectrometry. To identify the cross-linking sites, the ACR-treated peptide was digested with a protease and the resulting peptides were analysed by liquid chromatography-tandem mass spectrometry. Inter- and intra-molecular cross-linking adducts were identified between amino groups and the side chain of histidine in the peptide. These results indicated that the ACR-induced cross-links were accompanied by two reactions, namely Michael addition and Schiff base formation. In conclusion, the use of mass spectrometric techniques provided chemical evidence for protein cross-linking with ACR.

Can high-performance liquid chromatography coupled with fluorescence detection under all conditions be regarded as a sufficiently conclusive confirmatory method for B-group substances?

Commission Decision 2002/657/EC requires confirmatory analysis of B-group compounds when detected at levels above the permitted limit. In contrast to banned substances, for B-group substances, the use of mass spectrometric techniques is not obligatory and several techniques including liquid chromatography (LC)-ultraviolet light (UV) on two different LC columns and (single-column) high-performance liquid chromatography (HPLC)-fluorescence (Flu) are considered to deliver sufficient evidence for the identification of the detected substance. The analysis of sodium salicylate in animal drinking water collected at poultry farms is presented here as an example to show that even in a simple matrix such as animal drinking water, fluorescence detection in some cases may provide inadequate specificity. Of 50 samples analysed by LC-Flu, 18 tested positive for sodium salicylate. However, only in one sample was the presence of the analyte confirmed with mass spectrometric detection; the others were blank. Consequently, the LC-Flu results obtained were false-non-compliant for sodium salicylate. A second case concerning the analysis of avermectins in milk by HLPC-Flu is briefly described. For a number of samples analysed in the framework of a proficiency test, false non-compliant results for emamectin were reported due to a background interference sometimes present that practically co-eluted with the analyte. The observed retention time difference (1%) was well below the criterion (2.5%) specified in Commission Decision 2002/657/EC. Considering the impact of positive findings on individual farmers as well as on trade, product image and food safety perception by the consumer, it is concluded that also for B-group substances false-non-compliant results should be avoided whenever possible. This is especially important when the results are treated as and are expected to have the same repercussions as in the case of banned A-group substances. In these circumstances, only results obtained by mass spectrometry should be considered for confirmatory purposes.

Mass-spectrometric studies of C 3D 7NO molecule ionization by electron impact

New data on the measurements of ionization and dissociation ionization cross sections for deuterated dimethylformamide (C 3D 7NO) molecule by low-energy (0–100 eV) electron impact are presented. The use of mass-spectrometric technique has allowed the energy dependences of C 3D 7NO +, C 2D 2 +, C 2D 3 +, C 2D 4 +, C 2D 6N +, CDO +/C 3D 7NO ion yield cross sections to be obtained for the first time. The appearance energies for most of the ionized fragments studied have been determined by using the least-square fitting procedure.

Characterization of Pt Nanoparticles Encapsulated in Al2O3 and Their Catalytic Efficiency in Propene Hydrogenation

Pt nanoparticles supported in nanoporous Al2O3 catalyst are prepared by reduction of K2PtCl4 solution using H2 in the presence of Al2O3 and poly(acrylic acid) as capping material. After thorough washing with water to remove Pt nanoparticles located on the external surface of the Al2O3 and drying at 70 °C for 12 h, they were used in propene hydrogenation to evaluate catalytic activity as measured by the value of the activation energy in the temperature range between 30 and 90 °C. The Pt nanoparticles are characterized by using transmission electron microscopy (TEM). The particles in Pt/Al2O3 are found to be encapsulated and uniformly dispersed inside the Al2O3; however, the size and shapes are not clearly seen. After extraction of the Pt nanoparticles from the Al2O3 channels by using an ethanol-diluted HF solution, various shapes such as truncated octahedral, cubic, tetrahedral, and spherical with a size around 5 nm are observed. The encapsulated particles have various shapes but are smaller in size than those prepared in K2PtCl4 solution with polyacrylate in the absence of Al2O3. Using FT-IR studies, the capping material initially used in Pt/Al2O3 is not found in the Al2O3 channels. This might be due to the fact that the polymer (average MW 2100) is too large to be accommodated within the Al2O3 pores. The nanopores of Al2O3 have several roles in the synthesis of these nanoparticles. It allows for uniform dispersion and encapsulation of Pt nanoparticles. It controls the Pt sizes with narrow distribution that is determined by the pore dimension (5.8 nm). It protects against metal particle aggregation and produces various shapes even in the absence of the capping material. Using these Pt nanoparticles, the catalysis of hydrogenation of propene gas was studied. The initial rates, reaction order, rate constants, and activation energy for the hydrogenation are determined by use of mass spectrometric techniques. The activation energy is found to be 5.7 kcal/mol, which is about one-half that previously reported for catalysis by Pt metal deposited in SiO2 and TiO2 synthesized by using H2PtCl6 and Pt(allyl)2 by impregnation method.

Estimation of myocardial blood flow heterogeneity by transorgan helium transport functions.

Following single breath inhalations of helium 49 simultaneous inlet-outlet pairs of helium dilution curves were recorded in seven dog experiments from the aortic and coronary sinus blood by use of mass spectrometric technique. After numeric deconvolution of the dilution curves the weighting function of tracer transit times was used for calculation of the mean myocardial blood flow per unit of tissue, which closely correlated with directly measured flow. Secondly, the degree of blood flow heterogeneity was estimated from the first moments of the weighting function of transit times and by compartmental analysis based on an inverse Laplace transform. In the intact heart the results suggest a rather constant dispersion of flows relative to the mean flow F in the order of sigma/F = 0.3. The apparent dispersion of flows is increased by alpha-adrenergic stimulation with pressure and volume load, particularly in a state of beta-blockade, a finding, which may be attributed to variations of intercapillary distances and to a heterogeneity of blood flow per unit of tissue.