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Methods in Molecular Biology (Clifton,... 2024A number of different approaches have been used for quantitative peptidomics. In this protocol, we describe the method in which peptides are reacted with formaldehyde...
A number of different approaches have been used for quantitative peptidomics. In this protocol, we describe the method in which peptides are reacted with formaldehyde and sodium cyanoborohydride, which converts primary and secondary amines into tertiary amines. By using different combinations of regular reagents, deuterated reagents (H), and reagents containing deuterium and C, it is possible to produce five isotopically distinct forms of the methylated peptides, which can be quantified by mass spectrometry. Peptides with free N-termini that are primary amines incorporate two methyl groups using this procedure, which differ by 2 Da for each of the five isotopic combinations. Peptides that contain unmodified lysine residues incorporate additional pairs of methyl groups, leading to larger mass differences between isotopic forms. The reagents are commercially available, relatively inexpensive, and chemically stable.
Topics: Amines; Peptides; Mass Spectrometry; Methylation; Proteomics
PubMed: 38549011
DOI: 10.1007/978-1-0716-3646-6_6 -
Amyloid : the International Journal of... Dec 2023Immunoglobulin light chain (LC) amyloidosis is a life-threatening disease complicated by vast numbers of patient-specific mutations. We explored 14 patient-derived and...
BACKGROUND
Immunoglobulin light chain (LC) amyloidosis is a life-threatening disease complicated by vast numbers of patient-specific mutations. We explored 14 patient-derived and engineered proteins related to κ1-family germline genes IGKVLD-33*01 and IGKVLD-39*01.
METHODS
Hydrogen-deuterium exchange mass spectrometry analysis of conformational dynamics in recombinant LCs and their fragments was integrated with studies of thermal stability, proteolytic susceptibility, amyloid formation and amyloidogenic sequence propensity. The results were mapped on the structures of native and fibrillary proteins.
RESULTS
Proteins from two κ1 subfamilies showed unexpected differences. Compared to their germline counterparts, amyloid LC related to IGKVLD-33*01 was less stable and formed amyloid faster, whereas amyloid LC related to IGKVLD-39*01 had similar stability and formed amyloid slower, suggesting different major factors influencing amyloidogenesis. In 33*01-related amyloid LC, these factors involved destabilization of the native structure and probable stabilization of amyloid. The atypical behavior of 39*01-related amyloid LC stemmed from increased dynamics/exposure of amyloidogenic segments in βC'V and βEV that could initiate aggregation and decreased dynamics/exposure near the Cys23-Cys88 disulfide.
CONCLUSIONS
The results suggest distinct amyloidogenic pathways for closely related LCs and point to the complementarity-defining regions CDR1 and CDR3, linked via the conserved internal disulfide, as key factors in amyloid formation.
Topics: Humans; Immunoglobulin Light Chains; Complementarity Determining Regions; Amyloidosis; Amyloid; Immunoglobulin Light-chain Amyloidosis; Amyloidogenic Proteins; Disulfides
PubMed: 37216473
DOI: 10.1080/13506129.2023.2212397 -
NeuroImage Aug 2023Deuterium metabolic imaging (DMI) and quantitative exchange label turnover (QELT) are novel MR spectroscopy techniques for non-invasive imaging of human brain glucose...
INTRODUCTION
Deuterium metabolic imaging (DMI) and quantitative exchange label turnover (QELT) are novel MR spectroscopy techniques for non-invasive imaging of human brain glucose and neurotransmitter metabolism with high clinical potential. Following oral or intravenous administration of non-ionizing [6,6'-H]-glucose, its uptake and synthesis of downstream metabolites can be mapped via direct or indirect detection of deuterium resonances using H MRSI (DMI) and H MRSI (QELT), respectively. The purpose of this study was to compare the dynamics of spatially resolved brain glucose metabolism, i.e., estimated concentration enrichment of deuterium labeled Glx (glutamate+glutamine) and Glc (glucose) acquired repeatedly in the same cohort of subjects using DMI at 7T and QELT at clinical 3T.
METHODS
Five volunteers (4 m/1f) were scanned in repeated sessions for 60 min after overnight fasting and 0.8 g/kg oral [6,6'-H]-glucose administration using time-resolved 3D H FID-MRSI with elliptical phase encoding at 7T and 3D H FID-MRSI with a non-Cartesian concentric ring trajectory readout at clinical 3T.
RESULTS
One hour after oral tracer administration regionally averaged deuterium labeled Glx concentrations and the dynamics were not significantly different over all participants between 7T H DMI and 3T H QELT data for GM (1.29±0.15 vs. 1.38±0.26 mM, p=0.65 & 21±3 vs. 26±3 µM/min, p=0.22) and WM (1.10±0.13 vs. 0.91±0.24 mM, p=0.34 & 19±2 vs. 17±3 µM/min, p=0.48). Also, the observed time constants of dynamic Glc data in GM (24±14 vs. 19±7 min, p=0.65) and WM (28±19 vs. 18±9 min, p=0.43) dominated regions showed no significant differences. Between individual H and H data points a weak to moderate negative correlation was observed for Glx concentrations in GM (r=-0.52, p<0.001), and WM (r=-0.3, p<0.001) dominated regions, while a strong negative correlation was observed for Glc data GM (r=-0.61, p<0.001) and WM (r=-0.70, p<0.001).
CONCLUSION
This study demonstrates that indirect detection of deuterium labeled compounds using H QELT MRSI at widely available clinical 3T without additional hardware is able to reproduce absolute concentration estimates of downstream glucose metabolites and the dynamics of glucose uptake compared to H DMI data acquired at 7T. This suggests significant potential for widespread application in clinical settings especially in environments with limited access to ultra-high field scanners and dedicated RF hardware.
Topics: Humans; Magnetic Resonance Imaging; Deuterium; Reproducibility of Results; Brain; Glucose
PubMed: 37414233
DOI: 10.1016/j.neuroimage.2023.120250 -
Frontiers in Cellular Neuroscience 2024
PubMed: 38601024
DOI: 10.3389/fncel.2024.1401687 -
Journal of Labelled Compounds &... Apr 2024Kainate receptors play a crucial role in mediating synaptic transmission within the central nervous system. However, the lack of selective pharmacological tool compounds...
Kainate receptors play a crucial role in mediating synaptic transmission within the central nervous system. However, the lack of selective pharmacological tool compounds for the GluK3 subunit represents a significant challenge in studying these receptors. Recently presented compound 1 stands out as a potent antagonist of GluK3 receptors, exhibiting nanomolar affinity at GluK3 receptors and strongly inhibiting glutamate-induced currents at homomeric GluK1 and GluK3 receptors in HEK293 cells with K values of 65 and 39 nM, respectively. This study presents the synthesis of two potent GluK3-preferring iodine derivatives of compound 1, serving as precursors for radiolabelling. Furthermore, we demonstrate the optimisation of dehalogenation conditions using hydrogen and deuterium, resulting in [H]-1, and demonstrate the efficient synthesis of the radioligand [H]-1 with a specific activity of 1.48 TBq/mmol (40.1 Ci/mmol). Radioligand binding studies conducted with [H]-1 as a radiotracer at GluK1, GluK2, and GluK3 receptors expressed in Sf9 and rat P2 membranes demonstrated its potential applicability for selectively studying native GluK3 receptors in the presence of GluK1 and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-blocking ligands.
Topics: Rats; Animals; Humans; Tritium; Deuterium; HEK293 Cells; Receptors, Kainic Acid; Glutamic Acid; Receptors, AMPA
PubMed: 38332677
DOI: 10.1002/jlcr.4087 -
Infection and Drug Resistance 2023Antibiotic resistance represents a serious global health challenge, particularly with the emergence of strains resistant to last-resort antibiotics such as tigecycline,...
BACKGROUND
Antibiotic resistance represents a serious global health challenge, particularly with the emergence of strains resistant to last-resort antibiotics such as tigecycline, polymyxin B, and vancomycin. Urgent measures are required to alleviate this situation. To facilitate the judicious use of antibiotics, rapid and precise antimicrobial susceptibility testing (AST) is essential. Heavy water (deuterium oxide, DO)-labeled Raman spectroscopy has emerged as a promising time-saving tool for microbiological testing.
METHODS
Deuterium incorporation and experimental conditions were examined to develop and apply a Raman-based AST method to evaluate the efficacy of last-resort antibiotics, including tigecycline, polymyxin B, and vancomycin, against , and . Essential agreement and categorical agreement were used to assess the metabolism inactivation concentration based on Raman spectroscopy (R-MIC)-a new metric developed in this study-and minimum inhibitory concentration (MIC) determined via the traditional microdilution broth method. Spearman's rank correlation coefficient was employed to measure the association between R-MIC and MIC values.
RESULTS
The Raman-based AST method achieved a 100% categorical agreement (92/92) with the traditional microdilution broth method within five hours, while the traditional method required approximately 24 h. The R-MIC values shared 68.5% (63/92) consistency with the MIC values. In addition, the R-MIC and MIC values were highly correlated (Spearman's r=0.96), resulting in an essential agreement of 100%.
CONCLUSION
Our optimized experimental method and conditions indicate that Raman-based AST holds great promise as a solution to overcome the time-consuming challenges of traditional AST methods.
PubMed: 37638072
DOI: 10.2147/IDR.S404732 -
NMR in Biomedicine Aug 2023Deuterium metabolic imaging (DMI) is a novel noninvasive method to assess tissue metabolism and organ (patho)physiology in vivo using deuterated substrates, such as...
Deuterium metabolic imaging (DMI) is a novel noninvasive method to assess tissue metabolism and organ (patho)physiology in vivo using deuterated substrates, such as [6,6'- H ]-glucose. The liver and kidneys play a central role in whole-body glucose homeostasis, and in type 2 diabetes, both hepatic and renal glucose metabolism are dysregulated. Diabetes is also associated with gastric emptying abnormalities. In this study, we developed a four-channel H transmit/receive body array coil for DMI in the human abdomen at 7 T and assessed its performance. In addition, the feasibility of simultaneously measuring gastric emptying, and hepatic and renal glucose uptake and metabolism with dynamic 3D DMI upon administration of deuterated glucose, was investigated. Simulated and measured B patterns were in good agreement. The intrasession variability of the natural abundance deuterated water signal in the liver and right kidney, measured in nine healthy volunteers, was 5.6% ± 0.9% and 4.9% ± 0.7%, respectively. Dynamic 3D DMI scans with oral administration of [6,6'- H ]-glucose showed similar kinetics of deuterated glucose appearance and disappearance in the liver and kidney. The measured gastric emptying half time was 80 ± 10 min, which is in good agreement with scintigraphy measurements. In conclusion, DMI with oral administration of [6,6'- H ]-glucose enables simultaneous assessment of gastric emptying and liver and kidney glucose uptake and metabolism. When applied in patients with diabetes, this approach may advance our understanding of the interplay between disturbances in liver and kidney glucose uptake and metabolism and gastric emptying, at a detail that cannot be achieved by any other method.
Topics: Humans; Glucose; Gastric Emptying; Deuterium; Diabetes Mellitus, Type 2; Liver; Kidney
PubMed: 36929629
DOI: 10.1002/nbm.4926 -
Plant Methods Jul 2023Investigations into the growth and self-organization of plant roots is subject to fundamental and applied research in various areas such as botany, agriculture, and soil...
BACKGROUND
Investigations into the growth and self-organization of plant roots is subject to fundamental and applied research in various areas such as botany, agriculture, and soil science. The growth activity of the plant tissue can be investigated by isotope labeling experiments with heavy water and subsequent detection of the deuterium in non-exchangeable positions incorporated into the plant biomass. Commonly used analytical methods to detect deuterium in plants are based on mass-spectrometry or neutron-scattering and they either suffer from elaborated sample preparation, destruction of the sample during analysis, or low spatial resolution. Confocal Raman micro-spectroscopy (CRM) can be considered a promising method to overcome the aforementioned challenges. The substitution of hydrogen with deuterium results in the measurable shift of the CH-related Raman bands. By employing correlative approaches with a high-resolution technique, such as helium ion microscopy (HIM), additional structural information can be added to CRM isotope maps and spatial resolution can be further increased. For that, it is necessary to develop a comprehensive workflow from sample preparation to data processing.
RESULTS
A workflow to prepare and analyze roots of hydroponically grown and deuterium labeled Zea mays by correlative HIM-CRM micro-analysis was developed. The accuracy and linearity of deuterium detection by CRM were tested and confirmed with samples of deuterated glucose. A set of root samples taken from deuterated Zea mays in a time-series experiment was used to test the entire workflow. The deuterium content in the roots measured by CRM was close to the values obtained by isotope-ratio mass spectrometry. As expected, root tips being the most actively growing root zone had incorporated the highest amount of deuterium which increased with increasing time of labeling. Furthermore, correlative HIM-CRM analysis allowed for obtaining the spatial distribution pattern of deuterium and lignin in root cross-sections. Here, more active root zones with higher deuterium incorporation showed less lignification.
CONCLUSIONS
We demonstrated that CRM in combination with deuterium labeling can be an alternative and reliable tool for the analysis of plant growth. This approach together with the developed workflow has the potential to be extended to complex systems such as plant roots grown in soil.
PubMed: 37452400
DOI: 10.1186/s13007-023-01040-y -
Magnetic Resonance in Medicine Sep 2023To demonstrate the feasibility of deuterium echo-planar spectroscopic imaging (EPSI) to accelerate 3D deuterium metabolic imaging in the human liver at 7 T.
PURPOSE
To demonstrate the feasibility of deuterium echo-planar spectroscopic imaging (EPSI) to accelerate 3D deuterium metabolic imaging in the human liver at 7 T.
METHODS
A deuterium EPSI sequence, featuring a Hamming-weighted k-space acquisition pattern for the phase-encoding directions, was implemented. Three-dimensional deuterium EPSI and conventional MRSI were performed on a water/acetone phantom and in vivo in the human liver at natural abundance. Moreover, in vivo deuterium EPSI measurements were acquired after oral administration of deuterated glucose. The effect of acquisition time on SNR was evaluated by retrospectively reducing the number of averages.
RESULTS
The SNR of natural abundance deuterated water signal in deuterium EPSI was 6.5% and 5.9% lower than that of MRSI in the phantom and in vivo experiments, respectively. In return, the acquisition time of in vivo EPSI data could be reduced retrospectively to 2 min, beyond the minimal acquisition time of conventional MRSI (of 20 min in this case), while still leaving sufficient SNR. Three-dimensional deuterium EPSI, after administration of deuterated glucose, enabled monitoring of hepatic glucose dynamics with full liver coverage, a spatial resolution of 20 mm isotropic, and a temporal resolution of 9 min 50 s, which could retrospectively be shortened to 2 min.
CONCLUSION
In this work, we demonstrate the feasibility of accelerated 3D deuterium metabolic imaging of the human liver using deuterium EPSI. The acceleration obtained with EPSI can be used to increase temporal and/or spatial resolution, which will be valuable to study tissue metabolism of deuterated compounds over time.
Topics: Humans; Deuterium; Retrospective Studies; Echo-Planar Imaging; Magnetic Resonance Spectroscopy; Liver; Brain
PubMed: 37154391
DOI: 10.1002/mrm.29696 -
BioRxiv : the Preprint Server For... Jul 2023The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often...
The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium-exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.
PubMed: 37292983
DOI: 10.1101/2023.05.22.541822