The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a powerful tool for bioconjugation of biomolecules, particularly proteins and peptides. The major drawback limiting the use of the CuAAC reaction in biological systems is the copper-mediated formation of reactive oxygen species (ROS), leading to the oxidative degradation of proteins or peptides. From the studies on a limited number of proteins and peptides, it is known that, in general, the copper mediated oxidative damage is associated with the copper coordination environment and solvent accessibility. However, there is a lack of data to help estimate the extent of copper-mediated oxidation on a wide range of proteins and peptides. To begin to address this need, we quantitatively measured the degree of copper-mediated oxidation on libraries of 1200 tetrapeptides and a model protein (bovine serum albumin, BSA) using liquid chromatography mass spectrometry (LC-MS). The collected data will be useful to researchers planning to use the CuAAC reaction for bioconjugaton on peptides or proteins.
Background and PurposeNuclear magnetic resonance (NMR) spectroscopy has become an important technique for tissue studies. Since tissues are in semisolid-state, their high-resolution (HR) spectra cannot be obtained by conventional NMR spectroscopy. Because of this restriction, extraction and high-resolution magic angle spinning (HR MAS) are widely applied for HR NMR spectra of tissues. However, both of the methods are subject to limitations. In this study, the feasibility of HR 1H NMR spectroscopy based on intermolecular multiple-quantum coherence (iMQC) technique is explored using fish muscle, fish eggs, and a whole fish as examples.Materials and MethodsIntact salmon muscle tissues, intact eggs from shishamo smelt and a whole fish (Siamese algae eater) are studied by using conventional 1D one-pulse sequence, Hadamard-encoded iMQC sequence, and HR MAS.ResultsWhen we use the conventional 1D one-pulse sequence, hardly any useful spectral information can be obtained due to the severe field inhomogeneity. By contrast, HR NMR spectra can be obtained in a short period of time by using the Hadamard-encoded iMQC method without shimming. Most signals from fatty acids and small metabolites can be observed. Compared to HR MAS, the iMQC method is non-invasive, but the resolution and the sensitivity of resulting spectra are not as high as those of HR MAS spectra.ConclusionDue to the immunity to field inhomogeneity, the iMQC technique can be a proper supplement to HR MAS, and it provides an alternative for the investigation in cases with field distortions and with samples unsuitable for spinning. The acquisition time of the proposed method is greatly reduced by introduction of the Hadamard-encoded technique, in comparison with that of conventional iMQC method.
Phosphorus metabolism and circulation are essential bio-physicochemical processes during development of a plant and have been extensively studied and known to be affected by temperature, humidity, lighting, hormones etc. However, a quantitative description of how various phosphorous species evolve over time has not been reported. In this work, a combined 31P liquid and solid state NMR spectroscopic methodology is employed, supported by a new extraction scheme and data analysis method, to carry out a quantitative investigation of phosphorous circulation in germinating sesame seeds in dark and under illumination with and without adding a growth hormone. The spectra show that only slight changes occur for phosphorous metabolism at the initial stage but a rapid change takes place between 48–96 hours after germination is started. The metabolism is found to be temperature dependent and affected by illumination and hormone. However, neither illumination nor hormone affects the final residual concentration of phytin. Moreover, phytin does not flow out of cotyledon and the phosphorous flowing to other parts of the plant is always in the inorganic form. The overall evolution profile of phytate consumption is found to be a Gaussian decaying function. These findings can be explained with a dynamic model on phytin conversion.
The classification of Wuyi rock tea (Oolong type) is performed by solid‐state nuclear magnetic resonance (NMR) for the first time. Quality differences of eight Oolong teas grown in Wuyi Mountains in southeastern China are analyzed by a combination of solid‐state
1H and
1H– 13C CP‐MAS (cross‐polarization magic angle spinning) NMR, partial least‐squares discriminate analysis (PLS‐DA), and quantitative descriptive analysis (QDA). The contents of caffeine, carbohydrate, polyphenol, and terpenoid were distinguished, and quantification of the metabolites was made based on the
1H and
13C MAS spectra. PLS‐DA shows a separation of high‐ and low‐quality Oolong teas, in good agreement with the result of QDA. These results indicate that NMR metabolomic approach enables us to differentiate the analyzed teas based on their chemical composition.
Contrast enhancement agents are often employed in magnetic resonance imaging (MRI) for clinical diagnosis and biomedical research. However, the current theory on MRI contrast generation does not consider the ubiquitous presence of macromolecular crowders in biological systems, which poses the risk of inaccurate data interpretation and misdiagnosis. To address this issue, herein the macromolecular crowding effects on MRI contrast agent are investigated with the 1H relaxation rate of water in aqueous solutions of Dotarem with different concentrations of macromolecules. Two representative macromolecular crowder systems are used: polyethylene glycol (with no specific secondary structure) and bovine serum albumin (with compact secondary and tertiary structures). The water 1H relaxation rates in various solutions are measured in a fixed magnetic field and in variable magnetic fields. The results show significant crowding effects for both crowders. The relaxation rate is proportional to the concentration of the MRI contrast agent but shows conspicuous superlinearity with respect to the concentration of the crowder. The size of polyethylene glycol does not affect the relaxivity of water in Dotarem solutions. The above effects are verified with T
1‐ and T
2‐weighted NMR microimages. These results highlight the importance of the effect of macromolecular crowding on the MRI contrast agent and are valuable for understanding the mechanism of MRI contrast agents and designing new‐generation MRI contrast agents.
Intermolecular double quantum coherence (iDQC)1 H-nuclear magnetic resonance spectroscopy ( 1 H NMR), which serves as a complementary method in the analyses of fatty acids, is used to investigate the intact salmon muscle and the whole zebra fish. The spectra of fatty acids of the intact salmon muscle, whose resonances are overlapped with metabolites peaks in the conventional NMR spectra, can be resolved in the presence of severe intrinsic structural inhomogeneity without sample pretreatments and special NMR accessories, such as those for high-speed sample spinning. For improving the practicability of the iDQC method, a localized module is combined with the iDQC method so that the fatty acids of the whole zebra fish can be detected non-invasively. All the iDQC results are verified by the extraction NMR spectra. In addition, fatty acid composition of the salmon muscle is quantitatively analyzed based on the iDQC and extraction NMR spectra. The calculated results from these two methods are in good agreement. Therefore, the iDQC method may serve as a feasible one-step and fast screening method for fish quality analyses and lipid inspections of other biological tissues.Practical applications: The intermolecular doublequantum coherence (iDQC) can be an appropriate lipid detecting method of tissues when the samples are not suitable for chemical extraction. The iDQC with a localized module may be more practical than magic angle spinning for applications in in vivo and in situ NMR experiments, in which samples are not allowed to spin.
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