Project description:<p><strong>INTRODUCTION:</strong> The absolute quantitation of lipids at the lipidome-wide scale is a challenge but plays an important role in the comprehensive study of lipid metabolism. </p><p><strong>OBJECTIVES:</strong> We aim to develop a high-throughput quantitative lipidomics approach to enable the simultaneous identification and absolute quantification of hundreds of lipids in a single experiment. Then, we will systematically characterize lipidome-wide changes in the aging mouse brain and provide a link between aging and disordered lipid homeostasis. </p><p><strong>METHODS:</strong> We created an in-house lipid spectral library, containing 76,361 lipids and 181,300 MS/MS spectra in total, to support accurate lipid identification. Then, we developed a response factor-based approach for the large-scale absolute quantifications of lipids. </p><p><strong>RESULTS:</strong> Using the lipidomics approach, we absolutely quantified 1212 and 864 lipids in human cells and mouse brains, respectively. The quantification accuracy was validated using the traditional approach with a median relative error of 12.6%. We further characterized the lipidome-wide changes in aging mouse brains, and dramatic changes were observed in both glycerophospholipids and sphingolipids. Sphingolipids with longer acyl chains tend to accumulate in aging brains. Membrane-esterified fatty acids demonstrated diverse changes with aging, while most polyunsaturated fatty acids consistently decreased. </p><p><strong>CONCLUSION:</strong> We developed a high-throughput quantitative lipidomics approach and systematically characterized the lipidome-wide changes in aging mouse brains. The results proved a link between aging and disordered lipid homeostasis. </p><p><br></p><p><strong>Mouse brain NEG UPLC-MS assay</strong> data is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS495' rel='noopener noreferrer' target='_blank'><strong>MTBLS495</strong></a>.</p><p><strong>Mouse brain POS UPLC-MS assay</strong> data associated to this study is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS562' rel='noopener noreferrer' target='_blank'><strong>MTBLS562</strong></a>.</p>
Project description:<p><strong>INTRODUCTION:</strong> The absolute quantitation of lipids at the lipidome-wide scale is a challenge but plays an important role in the comprehensive study of lipid metabolism. </p><p><strong>OBJECTIVES:</strong> We aim to develop a high-throughput quantitative lipidomics approach to enable the simultaneous identification and absolute quantification of hundreds of lipids in a single experiment. Then, we will systematically characterize lipidome-wide changes in the aging mouse brain and provide a link between aging and disordered lipid homeostasis. </p><p><strong>METHODS:</strong> We created an in-house lipid spectral library, containing 76,361 lipids and 181,300 MS/MS spectra in total, to support accurate lipid identification. Then, we developed a response factor-based approach for the large-scale absolute quantifications of lipids. </p><p><strong>RESULTS:</strong> Using the lipidomics approach, we absolutely quantified 1212 and 864 lipids in human cells and mouse brains, respectively. The quantification accuracy was validated using the traditional approach with a median relative error of 12.6%. We further characterized the lipidome-wide changes in aging mouse brains, and dramatic changes were observed in both glycerophospholipids and sphingolipids. Sphingolipids with longer acyl chains tend to accumulate in aging brains. Membrane-esterified fatty acids demonstrated diverse changes with aging, while most polyunsaturated fatty acids consistently decreased. </p><p><strong>CONCLUSION:</strong> We developed a high-throughput quantitative lipidomics approach and systematically characterized the lipidome-wide changes in aging mouse brains. The results proved a link between aging and disordered lipid homeostasis. </p><p><br></p><p><strong>Mouse brain POS UPLC-MS assay</strong> data is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS562' rel='noopener noreferrer' target='_blank'><strong>MTBLS562</strong></a>. </p><p><strong>Mouse brain NEG UPLC-MS assay</strong> data associated to this study is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS495' rel='noopener noreferrer' target='_blank'><strong>MTBLS495</strong></a>.</p>
Project description:Aberrant sterol lipid metabolism is associated with physiological dysfunctions in the aging brain and aging-dependent disorders such as neurodegenerative diseases. There is an unmet demand to comprehensively profile sterol lipids spatially and temporally in different brain regions during aging. Here, we develop an ion mobility-mass spectrometry based four-dimensional sterolomics technology leveraged by a machine learning-empowered high-coverage library (>2000 sterol lipids) for accurate identification. We apply this four-dimensional technology to profile the spatially resolved landscapes of sterol lipids in ten functional regions of the mouse brain, and quantitatively uncover ~200 sterol lipids uniquely distributed in specific regions with concentrations spanning up to 8 orders of magnitude. Further spatial analysis pinpoints age-associated differences in region-specific sterol lipid metabolism, revealing changes in the numbers of altered sterol lipids, concentration variations, and age-dependent coregulation networks. These findings will contribute to our understanding of abnormal sterol lipid metabolism and its role in brain diseases.
Project description:We examined the effect of chronic high fat diet (HFD) on amyloid deposition and cognition of 12-months old APP23 mice, and correlated the phenotype to brain transcriptome and lipidome. HFD significantly increased amyloid plaques and worsened cognitive performance compared to mice on normal diet (ND).
Project description:Ion mobility can add a dimension to LC-MS based shotgun proteomics which has the potential to boost proteome coverage, quantification accuracy and dynamic range. Required for this is suitable software that extracts the information contained in the four-dimensional (4D) data space spanned by m/z, retention time, ion mobility and signal intensity. Here we describe the ion mobility enhanced MaxQuant software, which utilizes the added data dimension. It offers an end to end computational workflow for the identification and quantification of peptides, proteins and posttranslational modification sites in LC-IMS-MS/MS shotgun proteomics data. We apply it to trapped ion mobility spectrometry (TIMS) coupled to a quadrupole time-of-flight (QTOF) analyzer. A highly parallelizable 4D feature detection algorithm extracts peaks which are assembled to isotope patterns. Masses are recalibrated with a non-linear m/z, retention time, ion mobility and signal intensity dependent model, based on peptides from the sample. A new matching between runs (MBR) algorithm that utilizes collisional cross section (CCS) values of MS1 features in the matching process significantly gains specificity from the extra dimension. Prerequisite for using CCS values in MBR is a relative alignment of the ion mobility values between the runs. The missing value problem in protein quantification over many samples is greatly reduced by CCS aware MBR.MS1 level label-free quantification is also implemented which proves to be highly precise and accurate on a benchmark dataset with known ground truth. MaxQuant for LC-IMS-MS/MS is part of the basic MaxQuant release and can be downloaded from http://maxquant.org.
Project description:Brain-immune crosstalk and neuroinflammation critically shape brain physiology in health and disease. A detailed understanding of the brain immune landscape is essential for developing new treatments for many neurological disorders. Single-cell technologies offer an unbiased assessment of the heterogeneity, dynamics and functions of immune cells. Here, we provide a protocol that outlines all the steps involved for performing single-cell multi-omic analysis of the brain immune compartment. This includes a step-by-step description on how to micro-dissect the border regions of the mouse brain, together with dissociation protocols tailored to each of these tissues. These combine a high-yield with minimal dissociation-induced gene expression changes. Next, we outline the steps involved for droplet-based single-cell RNA sequencing (scRNA-seq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), or high-dimensional flow cytometry. Each of these single-cell modalities has specific strengths and limitations, but they are highly complementary. Importantly, we detail how to implement CITE-seq with large antibody panels to obtain unbiased protein-expression screening coupled to transcriptome analysis. Finally, we describe the main steps involved in the analysis and interpretation of the data. This optimized workflow allows for a detailed assessment of immune cell heterogeneity and activation in the whole brain or specific border regions, at RNA and protein level. The wetlab workflow can be completed by properly trained researchers (with basic proficiency in cell and molecular biology) and takes between 6 and 11 hours, depending on the chosen procedures. The computational analysis requires a background in bioinformatics and programming in R.
Project description:The biological significance of circular RNAs remains largely unexplored due to the lack of loss-of-function animal models. In this study, we focused on circTulp4, a highly abundant circRNA that is enriched in the brain and synaptic compartments. We created a circTulp4-deficient (CD) mouse model and conducted a comprehensive phenotypic analysis. To test whether the electrophysiological phenotype observed in CD mice was explained by an alteration in the levels of synaptic proteins, we prepared pure synaptosomal fractions and conducted a quantitative proteomic analysis. Label-free quantification of CD and wildtype synaptic proteomes was performed by using an ion mobility-enhanced data-independent acquisition (DIA) workflow with alternating low and elevated energy (referred to as UDMSE). We quantified >1800 proteins and concluded that the protein composition of the synaptosomal compartment is preserved in CD mice.
Project description:Maldi imaging with NEDC matrix of a rat brain tissue section. Image was acquired with 50 um resolution. Ion mobility seperation enabled. Negative ion mode.
Project description:22 Normal adult mouse tissues on custom alternative transcript sensitive Affymetrix microarray used to address differeneces in tissue specific alternative splicing. Abstract: Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe expression of RNA containing at least two alternative splice junctions for about a third of the 3200 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues, and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif strikingly similar to the branchpoint consensus, but which is located downstream of the 5' splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase (MAGUK) genes reveals that each contains a paralogous tissue regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families. Keywords: Alternative splicing isoform specific, adult mouse tissues