pure-shift-nuclear-magnetic-resonance-new-tool-for-plant

Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not ...

Pontificia Universidad Católica del Perú

Under physiological conditions, studies of intrinsically disordered proteins (IDPs) by conventional NMR methods based on proton detection are severely limited by fast amide-proton exchange with water. (13) C detection has been proposed as a solution to the exchange problem, but is hampered by low sensitivity. We propose a new pulse sequence combining proton-nitrogen cross-polarization and carbonyl detection to record high-resolution, high-sensitivity NMR spectra of IDPs under physiological conditions. To demonstrate the efficacy of this approach, we recorded a high-quality N-CO correlation spectrum of α-synuclein in bacterial cells at 37 °C.

Studying Intrinsically Disordered Proteins under True In Vivo Conditions by Combined Cross-Polarization and Carbonyl-Detection NMR Spectroscopy.

In this review, we focus on what we have learned from Nuclear Magnetic Resonance (NMR) studies on the neuronal microtubule-associated protein Tau. We consider both the mechanistic details of Tau: the tubulin relationship and its aggregation process. Phosphorylation of Tau is intimately linked to both aspects. NMR spectroscopy has depicted accurate phosphorylation patterns by different kinases, and its non-destructive character has allowed functional assays with the same samples. Finally, we will discuss other post-translational modifications of Tau and its interaction with other cellular factors in relationship to its (dys)function.

NMR Meets Tau: Insights into Its Function and Pathology.

The berry of Physalis peruviana L. (Solanaceae) represents an important socio-economical commodity for Latin America. The absence of a clear phenotype renders it difficult to trace its place of origin. In this study, Cape gooseberries from eight different regions within the Peruvian Andes were profiled for their metabolism implementing a NMR platform. Twenty-four compounds could be unequivocally identified and sixteen quantified. One-way ANOVA and post-hoc Tukey test revealed that all of the quantified metabolites changed significantly among regions: Bambamarca I showed the most accumulated significant differences. The coefficient of variation demonstrated high phenotypic plasticity for amino acids, while sugars displayed low phenotypic plasticity. Correlation analysis highlighted the closely coordinated behavior of the amino acid profile. Finally, PLS-DA revealed a clear separation among the regions based on their metabolic profiles, accentuating the discriminatory capacity of NMR in establishing significant phytochemical differences between producing regions of the fruit of P. peruviana L.

NMR-based metabolic study of fruits of Physalis peruviana L. grown in eight different Peruvian ecosystems.

Chagas disease is a trypanosomiasis disease inflicted by Trypanosoma cruzi parasite. In Latin America, at least 10 million people are infected and annually, 10,000 casualties are deplored. Macrophage infectivity potentiator protein is one of the major virulence factors secreted by T. cruzi (TcMIP) in order to infect its host but little is known about its mechanism of action. Studies confer TcMIP an important role in the extracellular matrix transmigration and basal lamina penetration. Here, we report the backbone H, C, and N resonance assignment of TcMIP and the comparison of the secondary structure obtained against reported X-ray crystallography data.

Backbone chemical shift assignment of macrophage infectivity potentiator virulence factor of Trypanosoma cruzi.

Even though Pure Shift NMR methods have conveniently been used in the assessment of crowded spectra, they are not commonly applied to the analysis of metabolomics data. This paper exploits the recently published SAPPHIRE-PSYCHE methodology in the context of plant metabolome. We compare single pulse, PSYCHE, and SAPPHIRE-PSYCHE spectra obtained from aqueous extracts of Physalis peruviana fruits. STOCSY analysis with simplified SAPPHIRE-PSYCHE spectra of six types of Cape gooseberry was carried out and the results attained compared with classical STOCSY data. PLS coefficients analysis combined with 1D-STOCSY was performed in an effort to simplify biomarker identification. Several of the most compromised proton NMR signals associated with critical constituents of the plant mixture, such as amino acids, organic acids, and sugars, were more cleanly depicted and their inter and intra correlation better reveled by the Pure Shift methods. The simplified data allowed the identification of glutamic acid, a metabolite not observed in previous studies of Cape gooseberry due to heavy overlap of its NMR signals. Overall, the results attained indicated that Ultra-Clean Pure Shift spectra increase the performance of metabolomics data analysis such as STOCSY and multivariate coefficients analysis, and therefore represent a feasible and convenient additional tool available to metabolomics.

Ultra-Clean Pure Shift H-NMR applied to metabolomics profiling.

Pyrazinamide (PZA) is an antibiotic used in first- and second-line tuberculosis treatment regimens. Approximately 50% of multidrug-resistant tuberculosis and over 90% of extensively drug-resistant tuberculosis strains are also PZA resistant. Despite the key role played by PZA, its mechanisms of action are not yet fully understood. It has been postulated that pyrazinoic acid (POA), the hydrolyzed product of PZA, could inhibit trans-translation by binding to Ribosomal protein S1 (RpsA) and competing with tmRNA, the natural cofactor of RpsA. Subsequent data, however, indicate that these early findings resulted from experimental artifact. Hence, in this study we assess the capacity of POA to compete with tmRNA for RpsA. We evaluated RpsA wild type (WT), RpsA ∆A438, and RpsA ∆A438 variants with truncations towards the carboxy terminal end. Interactions were measured using Nuclear Magnetic Resonance spectroscopy (NMR), Isothermal Titration Calorimetry (ITC), Microscale Thermophoresis (MST), and Electrophoretic Mobility Shift Assay (EMSA). We found no measurable binding between POA and RpsA (WT or variants). This suggests that RpsA may not be involved in the mechanism of action of PZA in Mycobacterium tuberculosis, as previously thought. Interactions observed between tmRNA and RpsA WT, RpsA ∆A438, and each of the truncated variants of RpsA ∆A438, are reported.

Mycobacterium tuberculosis ribosomal protein S1 (RpsA) and variants with truncated C-terminal end show absence of interaction with pyrazinoic acid.

Potato (Solanum tuberosum L.) is one of the world's most important crops, but it is facing major challenges due to climatic changes. To investigate the effects of intermittent drought on the natural variability of plant morphology and tuber metabolism in a novel potato association panel comprising 258 varieties we performed an augmented block design field study under normal irrigation and under water-deficit and recovery conditions in Ica, Peru. All potato genotypes were profiled for 45 morphological traits and 42 central metabolites via nuclear magnetic resonance. Statistical tests and norm of reaction analysis revealed that the observed variations were trait specific, that is, genotypic versus environmental. Principal component analysis showed a separation of samples as a result of conditional changes. To explore the relational ties between morphological traits and metabolites, correlation-based network analysis was employed, constructing one network for normal irrigation and one network for water-recovery samples. Community detection and difference network analysis highlighted the differences between the two networks, revealing a significant correlational link between fumarate and plant vigor. A genome-wide association study was performed for each metabolic trait. Eleven single nucleotide polymorphism (SNP) markers were associated with fumarate. Gene Ontology analysis of quantitative trait loci regions associated with fumarate revealed an enrichment of genes regulating metabolic processes. Three of the 11 SNPs were located within genes, coding for a protein of unknown function, a RING domain protein and a zinc finger protein ZAT2. Our findings have important implications for future potato breeding regimes, especially in countries suffering from climate change.

Morphological and metabolic profiling of a tropical-adapted potato association panel subjected to water recovery treatment reveals new insights into plant vigor.

Chitosan is a biodegradable, antibacterial, and nontoxic biopolymer used in a wide range of applications including biotechnology, pharmacy, and medicine. The physicochemical and biological properties of chitosan have been associated with parameters such as the degree of polymerization (DP) and the fraction of acetylation (). New methods are being developed to yield chitosans of specific acetylation patterns, and, recently, a correlation between biological activity and the distribution of the acetylated units (: pattern of acetylation) has been demonstrated. Although there are numerous well-established methods for the determination of DP and  values, this is not the case for . The methods available are either not straightforward or not sensitive enough, limiting their use for routine analysis. In this study, we demonstrate that by applying HOmodecoupled Band-Selective (HOBS) decoupling NMR on signals assigned by multidimensional Pure Shift NMR methods,  can be easily and accurately determined on various chitosan samples. This novel methodology-easily implemented for routine analysis-could become a standard for chitosan  assessment. In addition, by applying Spectral Aliased Pure Shift HSQC, the analysis was enhanced with the determination of triads.

Study of the Acetylation Pattern of Chitosan by Pure Shift NMR.

The fruit of Vanilla planifolia is broadly preferred by the agroindustry and gourmet markets due to its refined flavor and aroma. Peruvian Vanilla has been proposed as a possible source for genetic improvement of existing Vanilla cultivars, but, little has been done to facilitate comprehensive studies of these and other Vanilla. Here, a nuclear magnetic resonance (NMR) metabolomic platform was developed to profile for the first time the leaves - organ known to accumulate vanillin putative precursors - of V. planifolia and those of Peruvian V. pompona, V. palmarum, and V. ribeiroi, with the aim to determine metabolic differences among them. Analysis of the NMR spectra allowed the identification of thirty-six metabolites, twenty-five of which were quantified. One-way ANOVA and post-hoc Tukey test revealed that these metabolites changed significantly among species, whilst multivariate-analyses allowed the identification of malic and homocitric acids, together with two vanillin precursors, as relevant metabolic markers for species differentiation.

Juan M. Lopez

Departamento de Ciencias - Química,
CERMN,
Departamento de Ciencias - Química, CERMN

Pontificia Universidad Católica del Perú

Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics

Juan M. Lopez

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Departamento de Ciencias - Química,CERMN,Departamento de Ciencias - Química, CERMN

Pontificia Universidad Católica del Perú

Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics

Departamento de Ciencias - Química,
CERMN,
Departamento de Ciencias - Química, CERMN