The “ForensOMICS” approach to forensic post-mortem interval estimation: combining metabolomics, lipidomics and proteomics for the analysis human skeletal remains
Project description:The combined use of multiple omics allows to study complex interrelated biological processes in their entirety. We applied a combination of metabolomics, lipidomics and proteomics to human bones to investigate their combined potential to estimate time elapsed since death (i.e., the postmortem interval [PMI]). This 'ForensOMICS' approach has the potential to improve accuracy and precision of PMI estimation of skeletonized human remains, thereby helping forensic investigators to establish the timeline of events surrounding death. Anterior midshaft tibial bone was collected from four female body donors before their placement at the Forensic Anthropology Research Facility owned by the Forensic Anthropological Center at Texas State (FACTS). Bone samples were again collected at selected PMIs (219-790-834-872days). Liquid chromatography mass spectrometry (LC-MS) was used to obtain untargeted metabolomic, lipidomic, and proteomic profiles from the pre- and post-placement bone samples. The three omics blocks were investigated independently by univariate and multivariate analyses, followed by Data Integration Analysis for Biomarker discovery using Latent variable approaches for Omics studies (DIABLO), to identify the reduced number of markers describing postmortem changes and discriminating the individuals based on their PMI. The resulting model showed that pre-placement metabolome, lipidome and proteome profiles were clearly distinguishable from post-placement ones. Metabolites in the pre-placement samples suggested an extinction of the energetic metabolism and a switch towards another source of fuelling (e.g., structural proteins). We were able to identify certain biomolecules with an excellent potential for PMI estimation, predominantly the biomolecules from the metabolomics block. Our findings suggest that, by targeting a combination of compounds with different postmortem stability, in the future we could be able to estimate both short PMIs, by using metabolites and lipids, and longer PMIs, by using proteins.
Project description:Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.
Project description:Given the limited accuracy of clinically used risk scores such as the Systematic COronary Risk Evaluation 2 system and the Second Manifestations of ARTerial disease 2 risk scores, novel risk algorithms determining an individual's susceptibility of future incident or recurrent atherosclerotic cardiovascular disease (ASCVD) risk are urgently needed. Due to major improvements in assay techniques, multimarker proteomic and lipidomic panels hold the promise to be reliably assessed in a high-throughput routine. Novel machine learning-based approaches have facilitated the use of this high-dimensional data resulting from these analyses for ASCVD risk prediction. More than a dozen of large-scale retrospective studies using different sets of biomarkers and different statistical methods have consistently demonstrated the additive prognostic value of these panels over traditionally used clinical risk scores. Prospective studies are needed to determine the clinical utility of a biomarker panel in clinical ASCVD risk stratification. When combined with the genetic predisposition captured with polygenic risk scores and the actual ASCVD phenotype observed with coronary artery imaging, proteomics and lipidomics can advance understanding of the complex multifactorial causes underlying an individual's ASCVD risk.
Project description:Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious requiring two hours of mass spectrometry time per single muscle fiber; 50 fibers would take approximately four days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 minutes total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 hours. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Sixty-five proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, muscle structure and regulation. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput.
Project description:This study investigates the impact of sodium channel protein type 1 subunit alpha (SCN1A) gene knockout (SCN1A KO) on brain development and function using cerebral organoids coupled with a multiomics approach. From comprehensive omics analyses, we found that SCN1A KO organoids exhibit decreased growth, dysregulated neurotransmitter levels, and altered lipidomic, proteomic, and transcriptomic profiles compared to controls under matrix-free differentiation conditions. Neurochemical analysis reveals reduced levels of key neurotransmitters, and lipidomic analysis highlights changes in ether phospholipids and sphingomyelin. Furthermore, quantitative profiling of the SCN1A KO organoid proteome shows perturbations in cholesterol metabolism and sodium ion transportation, potentially affecting synaptic transmission. These findings suggest dysregulation of cholesterol metabolism and sodium ion transport, with implications for synaptic transmission. Overall, these insights shed light on the molecular mechanisms underlying SCN1A-associated disorders, such as Dravet syndrome, and offer potential avenues for therapeutic intervention.
Project description:Accurate estimation of the postmortem interval (PMI) is crucial in forensic medico-legal investigations to understand case circumstances (e.g. narrowing down list of missing persons or include/exclude suspects). Due to the complex decomposition chemistry, estimation of PMI remains challenging and currently often relies on the subjective visual assessment of gross morphological/taphonomic changes of a body during decomposition or entomological data. The aim of the current study was to investigate the human decomposition process up to 3 months after death and propose novel time-dependent biomarkers (peptide ratios) for the estimation of decomposition time. An untargeted liquid chromatography tandem mass spectrometry-based bottom-up proteomics workflow (ion mobility separated) was utilized to analyse skeletal muscle, collected repeatedly from nine body donors decomposing in an open eucalypt woodland environment in Australia. Additionally, general analytical considerations for large-scale proteomics studies for PMI determination are raised and discussed. Multiple peptide ratios (human origin) were successfully proposed (subgroups < 200 accumulated degree days (ADD), < 655 ADD and < 1535 ADD) as a first step towards generalised, objective biochemical estimation of decomposition time. Furthermore, peptide ratios for donor-specific intrinsic factors (sex and body mass) were found. Search of peptide data against a bacterial database did not yield any results most likely due to the low abundance of bacterial proteins within the collected human biopsy samples. For comprehensive time-dependent modelling, increased donor number would be necessary along with targeted confirmation of proposed peptides. Overall, the presented results provide valuable information that aid in the understanding and estimation of the human decomposition processes.
Project description:Circadian clocks play an important role in lipid homeostasis, with impact on various metabolic diseases. Due to the central role of skeletal muscle in whole-body metabolism, we aimed at studying muscle lipid profiles in a temporal manner. Moreover, it has not been shown whether lipid oscillations in peripheral tissues are driven by diurnal cycles of rest-activity and food intake or are able to persist in vitro in a cell-autonomous manner. To address this, we investigated lipid profiles over 24 h in human skeletal muscle in vivo and in primary human myotubes cultured in vitro. Glycerolipids, glycerophospholipids, and sphingolipids exhibited diurnal oscillations, suggesting a widespread circadian impact on muscle lipid metabolism. Notably, peak levels of lipid accumulation were in phase coherence with core clock gene expression in vivo and in vitro. The percentage of oscillating lipid metabolites was comparable between muscle tissue and cultured myotubes, and temporal lipid profiles correlated with transcript profiles of genes implicated in their biosynthesis. Lipids enriched in the outer leaflet of the plasma membrane oscillated in a highly coordinated manner in vivo and in vitro. Lipid metabolite oscillations were strongly attenuated upon siRNA-mediated clock disruption in human primary myotubes. Taken together, our data suggest an essential role for endogenous cell-autonomous human skeletal muscle oscillators in regulating lipid metabolism independent of external synchronizers, such as physical activity or food intake.
Project description:Trans fatty acid consumption in the human diet can cause adverse health effects, such as cardiovascular disease, which is associated with higher total cholesterol, a higher low density lipoprotein-cholesterol level and a decreased high density lipoprotein-cholesterol level. The aim of the study was to elucidate the hepatic response to the most abundant trans fatty acid in the human diet, elaidic acid, to help explain clinical findings on the relationship between trans fatty acids and cardiovascular disease. The human HepG2 cell line was used as a model to investigate the hepatic response to elaidic acid in a combined proteomic, transcriptomic and lipidomic approach. We found many of the proteins responsible for cholesterol synthesis up-regulated together with several proteins involved in the esterification and hepatic import/export of cholesterol. Furthermore, a profound remodeling of the cellular membrane occurred at the phospholipid level. Our findings contribute to the explanation on how trans fatty acids from the diet can cause modifications in plasma cholesterol levels by inducing abundance changes in several hepatic proteins and the hepatic membrane composition.
Project description:Post-translational modifications (PTMs) (e.g., acetylation, methylation, and phosphorylation) play crucial roles in regulating the diverse protein-protein interactions involved in essentially every cellular process. While significant progress has been made to detect PTMs, profiling protein-protein interactions mediated by these PTMs remains a challenge. Here, we report a method that combines a photo-cross-linking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein-protein interactions. To develop and apply this approach, we focused on trimethylated lysine-4 at the histone H3 N-terminus (H3K4Me(3)), a PTM linked to actively transcribed gene promoters. Our approach identified proteins previously known to recognize this modification and MORC3 as a new protein that binds H3M4Me(3). This study indicates that our cross-linking-assisted and SILAC-based protein identification (CLASPI) approach can be used to profile protein-protein interactions mediated by PTMs, such as lysine methylation.
Project description:Salivary glands provide secretory functions, including secretion of xenobiotics and among them drugs. However, there is no published information about protein abundance of drug transporters measured using reliable protein quantification methods. Therefore, mRNA expression and absolute protein content of clinically relevant ABC (n = 6) and SLC (n = 15) family member transporters in the human parotid gland, using the qRT-PCR and liquid chromatography‒tandem mass spectrometry (LC-MS/MS) method, were studied. The abundance of nearly all measured proteins ranged between 0.04 and 0.45 pmol/mg (OCT3 > MRP1 > PEPT2 > MRP4 > MATE1 > BCRP). mRNAs of ABCB1, ABCC2, ABCC3, SLC10A1, SLC10A2, SLC22A1, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLCO1A2, SLCO1B1, SLCO1B3 and SLCO2B1 were not detected. The present study provides, for the first time, information about the protein abundance of membrane transporters in the human parotid gland, which could further be used to define salivary bidirectional transport (absorption and secretion) mechanisms of endogenous compounds and xenobiotics.