Project description:Human activities and climate change have negatively affected the world's oceans, leading to a 30-60% decline in biodiversity and habitats in coastal ecosystems. Marine turtles, as bioindicator species, accumulate contaminants, including trace elements, due to their extensive migration and long life span. However, there is a lack of data on the abundance of these contaminants and their effects on marine turtles' health. This study focuses on analyzing the muscle proteome of juvenile green sea turtles (Chelonia mydas) from Reunion Island. The ultimate goal was to evaluate whether muscle proteome responds to in-situ mixtures of inorganic contaminants to decipher the possible impacts on individual health, thereby identifying potential new biomarkers for long-term monitoring and conservation efforts.
Project description:The erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, was the first hematopoietic niche discovered. The identity of EBI macrophages has thus far remained elusive. Gene expression profiles of BM F4/80+Epor-eGFP+ macrophages suggest a specialized function in supporting erythropoiesis.
Project description:We performed a comprehensive fecal metabolite analysis using LC-MS/MS and LC-QTOF-MS approaches as a preliminary study. Feces of Japanese macaques on Yakushima Island were collected from five monkeys at two separate locations. Using the former methodology, 59 substances such as free amino acids, nucleotides, nucleosides and nucleic acid bases, and organic acids in the citrate cycle were quantitatively detected and successfully differentiated in two different monkey groups by the concentrations of nucleic acid metabolites and free amino acids. In the latter, around 12,000 substances were detected both by positive and negative mode in each sample. Differences in signal intensities were observed between two monkey groups in the concentrations of plant secondary metabolites such as cyanogenic glycosides, flavonoids, and phenolics.
Project description:In this study, both conventional one-dimensional liquid chromatography (1DLC) and comprehensive two-dimensional liquid chromatography (2DLC) coupled to a high-resolution time-of-flight mass spectrometer (HR-TOF MS) were used for full-scale lipid characterization of lipid extracts from zebrafish embryos. We investigated the influence on annotated lipids and different separation mechanisms (HILIC, C18, and PFP), and their different orders arranged in the first and the second dimensions. As a result, the number of lipid species annotated by conventional one-dimensional LC-MS was between 212 and 448. In contrast, the number of individual lipids species annotated by C18×HILIC, HILIC×C18, and HILIC×PFP were 1784, 1059, and 1123, respectively. Therefore, it was evident that the performance of comprehensive 2DLC, especially the C18×HILIC method, considerably exceeded 1DLC. Interestingly, a comparison of the HILIC×C18 and C18×HILIC approaches showed, under the optimized conditions, similar orthogonality, but the effective separation power of the C18×HILIC was much higher. A comparison of the HILIC×C18 and the HILIC×PFP methods demonstrated that the HILIC×PFP separation had superior orthogonality with a small increase on its effective peak capacity, indicating that the HILIC×PFP combination maybe a promising platform for untargeted lipidomics in complex samples. Finally, from the comprehensive lipid profiling respective, the C18×HILIC was selected for further studies.
Project description:We have developed new applications of the pseudocolor plot for the analysis of LC/MS data. These applications include spectral averaging, analysis of variance, differential comparison of spectra, and qualitative filtering by compound class. These applications have been motivated by the need to better understand LC/MS data generated from analysis of human biofluids. The examples presented use data generated to profile steroid hormones in urine extracts from a Cushing's disease patient relative to a healthy control, but are general to any discovery-based scanning mass spectrometry technique. In addition to new visualization techniques, we introduce a new metric of variance: the relative maximum difference from the mean. We also introduce the concept of substructure-dependent analysis of steroid hormones using precursor ion scans. These new analytical techniques provide an alternative approach to traditional untargeted metabolomics workflow. We present an approach to discovery using MS that essentially eliminates alignment or preprocessing of spectra. Moreover, we demonstrate the concept that untargeted metabolomics can be achieved using low mass resolution instrumentation.
Project description:Glycated proteins are emerging as good indicators for diabetes and age related diseases. However, the platform for analysis of glycated proteome has been relatively less well established. We here introduce an online 2D-LC-HCD-MS/MS platform for comprehensive glycated peptide quantification. This platform includes a boronate affinity column in the first dimension for enrichment, reversed phase nanoLC column in the second dimension for separation, a benchtop Orbitrap mass spectrometer with HCD-MS/MS for peptide sequencing, and MaxQuant bioinformatics tool for identification and quantification of glycated peptides. This online 2D-LC-HCD-MS/MS platform has high enrichment efficiency with 85% of identified peptides in the enriched fraction as glycated, high sensitivity for detection of glycated peptides with LOD and LOQ at 1.2 and 2.4 pg, respectively, and high reproducibility with interday CVs < 20% for 80% of the glycated peptides. The number of glycated peptides quantified in in vitro glycated human plasma increased more than 3-fold using this platform in comparison to that obtained using 1D-LC-HCD-MS/MS platform without boronate affinity enrichment. Application of this online platform to human plasma identified 376 glycated peptides from 10 ?g of protein digests. This highly sensitive and reproducible online 2D platform is promising for glycated protein analysis of complex clinical samples.
Project description:Toxicoproteomics is a developing field that utilizes global proteomic methodologies to investigate the physiological response as a result of adverse toxicant exposure. The aim of this study was to compare the protein secretion profile in lung bronchoalveolar lavage fluid (BALF) from mice exposed to non-functionalized multi-walled carbon nanotubes (U-MWCNTs) or MWCNTs functionalized by nanoscale Al2O3 coatings (A-MWCNT) formed using atomic layer deposition (ALD). Proteins were identified using liquid chromatography tandem mass spectrometry (LC-MS/MS), and quantified using a combination of two label-free proteomic methods: spectral counting and MS1 peak area analysis. On average 465 protein groups were identified per sample and proteins were first screened using spectral counting and the Fisher's exact test to determine differentially regulated species. Significant proteins by Fisher's exact test (p<0.05) were then verified by integrating the intensity under the extracted ion chromatogram from a single unique peptide for each protein across all runs. A two sample t-test based on integrated peak intensities discovered differences in 27 proteins for control versus U-MWCNT, 13 proteins for control versus A-MWCNT, and 2 proteins for U-MWCNT versus A-MWCNT. Finally, an in-vitro binding experiment was performed yielding 4 common proteins statistically different (p<0.05) for both the in-vitro and in-vivo study. Several of the proteins found to be significantly different between exposed and control groups are known to play a key role in inflammatory and immune response. A comparison between the in-vitro and in-vivo CNT exposure emphasized a true biological response to CNT exposure.