Schroederoverby2934
Inclusion lists were created automatically based on full scan MS signals from samples and through iterative analyses, ions in the inclusion list that were fragmented were automatically moved to the exclusion list in subsequent runs. We confirmed that analytes with low MS response that did not undergo MS/MS events in conventional data-dependent analysis were successfully fragmented using this approach. Overall, this automated data-driven data acquisition approach resulted in a higher coverage of lipidome and the use of dual dissociation techniques provided additional information that was critical in characterizing the side chains of phosphatidylcholine species.Large epidemiological studies often require sample transportation and storage, presenting unique considerations when applying advanced lipidomics techniques. The goal of this study was to acquire lipidomics data on plasma and serum samples stored at potential preanalytical conditions (e.g., thawing, extracting, evaporating), systematically monitoring lipid species for a period of one month. Split aliquots of 10 plasma samples and 10 serum samples from healthy individuals were kept in three temperature-related environments refrigerator, laboratory benchtop, or heated incubator. Samples were analyzed at six different time points over 28 days using a Bligh & Dyer lipid extraction protocol followed by direct infusion into a lipidomics platform using differential mobility with tandem mass spectrometry. The observed concentration changes over time were evaluated relative to method and inter-individual biological variability. In addition, to evaluate the effect of lipase enzyme levels on concentration changes during storage, we compared corresponding fasting and post-prandial plasma samples collected from 5 individuals. Based on our data, a series of low abundance free fatty acid (FFA), diacylglycerol (DAG), and cholesteryl ester (CE) species were identified as potential analytical markers for degradation. These FFA and DAG species are typically produced by endogenous lipases from numerous triacylglycerols (TAGs), and certain high abundance phosphatidylcholines (PCs). The low concentration CEs, which appeared to increase several fold, were likely mass-isobars from oxidation of other high concentration CEs. Although the concentration changes of the high abundant TAG, PC, and CE precursors remained within method variability, the concentration trends of FFA, DAG, and oxidized CE products should be systematically monitored over time to inform analysts about possible pre-analytical biases due to degradation in the study sample sets.Plasmalogens (Pls) levels are reported to be altered in several neurological and metabolic diseases. Identification of sn-1 fatty alcohols and sn-2 fatty acids of different Pls species is necessary to determine the roles and mechanisms of action of Pls in different diseases. Previously, full-scan tandem mass spectrometry (MS/MS) was used for this purpose but is not effective for low-abundance Pls species. Recently, multiplexed selected reaction monitoring MS (SRM/MS) was found to be more selective and sensitive than conventional full-scan MS/MS for the identification of low-abundance compounds. In the present study, we developed a liquid chromatography (LC)-targeted multiplexed SRM/MS system for the identification and quantification of different Pls choline (Pls-PC) and Pls ethanolamine (Pls-PE) species. We determined five precursor-product ion transitions to identify sn-1 and sn-2 fragments of each Pls species. Consequently, sn-1 and sn-2 fatty acyl chains of 22 Pls-PC and 55 Pls-PE species were identified in mouse brain samples. Among them, some species had C200 and C201 fatty alcohols at the sn-1 position. For quantification of Pls species in mouse brain samples, a single SRM transition was employed. Thus, our results suggest that the LC-targeted multiplexed SRM/MS system is very sensitive for the identification and quantification of low-abundance lipids such as Pls, and is thus expected to make a significant contribution to basic and clinical research in this field in the future.
The worldwide prevalence of non-alcoholic fatty liver disease (NAFLD) has stimulated work to identify biomarkers and develop effective treatments. Metabolomics is an emerging tool that has been widely applied to discover biomarkers and simultaneously uncover pathological mechanisms. Here, we aim to optimize metabolomic acquisition with the goal of obtaining a systemic metabolic profile to unravel the potential link between dysregulated metabolism and NAFLD.
We analyzed serum samples collected from healthy subjects (n=8) and NAFLD patients (n=8) via an integrative analytical workflow using two orthogonal separation modes with T3 and amide columns and two ionization polarity modes on a UPLC-ESI-Q/TOF. Data dependent acquisition was employed for data acquisition. Differentially expressed metabolites and lipids were identified by comparing the collected metabolic and lipidomic profiles between the healthy subjects and NAFLD patients.
The integrative LC-MS/MS analytical workflow employed here features an improved coverage of metabolites and lipids, which leads to the identification of 20 potential biomarkers of NAFLD, including lipids, acylcarnitines, and organic acids.
This pilot study has identified potential biomarkers for NAFLD and revealed corresponding dysregulated metabolic pathways related to NAFLD's occurrence and progression, establishing a molecular basis for NAFLD diagnosis and therapeutic intervention.
This pilot study has identified potential biomarkers for NAFLD and revealed corresponding dysregulated metabolic pathways related to NAFLD's occurrence and progression, establishing a molecular basis for NAFLD diagnosis and therapeutic intervention.
With the rising complexity of modern multimarker analytical techniques and notable scientific publication retractions required for erroneous statistical analysis, there is increasing awareness of the importance of research transparency and reproducibility. The development of mature open-source tools for literate programming in multiple langauge paradigms has made fully-reproducible authorship possible.
We describe the procedure for manuscript preparation using RMarkdown and the R statistical programming language with application to JMSACL or any other Elsevier journal.
An instructional manuscript has been prepared in the RMarkdown markup language with stepwise directions on preparing sections, subsections, lists, tables, figures and reference management in an entirely reproducible format.
From RMarkdown code, a submission-ready PDF is generated and JMSACL-compatible LaTeX code is generated. These can be uploaded to the Editorial Manager.
A completely reproducible manuscript preparation pipeline using the R and RMarkdown is described.
A completely reproducible manuscript preparation pipeline using the R and RMarkdown is described.Alcohol biomarkers can monitor both recent and long-term drinking and provide information about drinking habits as a complement to self-reporting. Ethyl glucuronide (EtG) and phosphatidylethanol (PEth) are the most sensitive available biomarkers for this purpose. The present study aimed to collect data on both PEth and EtG in the same blood sample, in addition to ethanol, in order to evaluate the combined use of these biomarkers. Venous EDTA blood samples (n = 1149) sent to the laboratory as part of a clinical routine service for measuring PEth were investigated. PEth and EtG concentrations were analyzed using liquid chromatography-mass spectrometry methods and ethanol with an enzymatic method. Of the 1149 samples, 95 were positive for ethanol (range 0.11-3.12 g/L), 454 for EtG (1.0-9739 ng/mL), 635 for PEth (0.014-6.0 µmol/L), 534 for PEth ≥ 0.050 µmol/L, and 315 for PEth ≥ 0.30 µmol/L. EtG and PEth concentrations seemed largely independent as the coefficient of determination (r2) between PEth and EtG concentrations was 0.15. However, when the EtG concentrations were evaluated for different subgroups depending on ethanol or PEth concentrations a statistically significant difference between successive higher concentrations was observed. EtG and PEth are independent measures of recent alcohol drinking reflecting different time windows. Their combined measurement in the same blood sample is possible and will provide valuable information regarding recent alcohol consumption as a complement to self-reporting.Understanding the plasma membrane nanoscale organization and dynamics in living cells requires microscopy techniques with high spatial and temporal resolution that permit for long acquisition times and allow for the quantification of membrane biophysical properties, such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolutions, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dye is showcased by live-cell real-time three-dimensional STED recordings of bleb formation and lipid exchange during membrane fusion as well as by STED-fluorescence correlation spectroscopy experiments for the simultaneous quantification of membrane dynamics and lipid packing that correlate in model and live-cell membranes.Mapping DNA damage and its repair has immense potential in understanding environmental exposures, their genotoxicity, and their impact on human health. Monitoring changes in genomic stability also aids in the diagnosis of numerous DNA-related diseases, such as cancer, and assists in monitoring their progression and prognosis. Developments in recent years have enabled unprecedented sensitivity in quantifying the global DNA damage dose in cells via fluorescence-based analysis down to the single-molecule level. However, genome-wide maps of DNA damage distribution are challenging to produce. Here, we describe the localization of DNA damage and repair loci by repair-assisted damage detection sequencing (RADD-seq). Based on the enrichment of damage lesions coupled with a pull-down assay and followed by next-generation sequencing, this method is easy to perform and can produce compelling results with minimal coverage. Selleckchem Aurora A Inhibitor I RADD-seq enables the localization of both DNA damage and repair sites for a wide range of single-strand damage types. Using this technique, we created a genome-wide map of the oxidation DNA damage lesion 8-oxo-7,8-dihydroguanine before and after repair. Oxidation lesions were heterogeneously distributed along the human genome, with less damage occurring in tight chromatin regions. Furthermore, we showed repair is prioritized for highly expressed, essential genes and in open chromatin regions. RADD-seq sheds light on cellular repair mechanisms and is capable of identifying genomic hotspots prone to mutation.
