A plethora of studies suggest that a high-fat diet in combination with a high amyloid load causes synaptic insulin resistance and is a risk factor for Alzheimer's disease. Our understanding of the underlying mechanisms is still fragmented. To gain new insights, we conducted integrated proteomic and phosphoproteomic profiling of hippocampal synaptosomes from WT and a transgenic mouse line with a high amyloid load (heterozygous TBA2.1 mice) that show no overt signs of neurodegeneration and dementia. Mice were fed with a regular or high-fat diet. Data-independent acquisition quantified over 5400 proteins, revealing a stable synaptic proteome across conditions. However, the combination of high amyloid load and high-fat diet triggered coordinated remodeling of lipid metabolism pathways, particularly mitochondrial and peroxisomal fatty acid catabolism. Phosphoproteomic analysis showed pronounced activation of lipid- and stress-responsive kinases, including protein kinase C-α, along with increased inhibitory phosphorylation of insulin receptor substrates (IRS1/2). In vitro experiments indicate that blocking protein kinase C-α indeed prevents synaptic insulin resistance in primary neurons. The findings suggest that this proteomic workflow, combined with kinase pathway analysis, can reveal nodal points for interventions in a complex disease state with a trajectory to Alzheimer's disease.

The rapid and continued development of mass spectrometry-based technologies has significantly increased the capability to study and characterize lipids while providing new insight into the complex roles of lipids throughout biology. These capabilities have included the ability to quantify, structurally characterize (including resolving isomers that pose significant challenges to lipidomics), and spatially map lipids within numerous complex organisms, revealing new capabilities in emerging areas such as single-cell analysis. With these rapid developments, several challenges have emerged, such as accurate lipid identification, incorrect and overinterpretation of mass spectrometry data and structural assignments, and the need for improved analytical and bioinformatics tools to understand lipidomics data at the pathway and systems levels. This review critically assesses analytical technologies used for lipidomics studies, along with current challenges and technological developments driving the field forward. By highlighting these challenges, and possible avenues to address them, this review emphasizes the excitement for the future of lipidomics and the need for continued development of analytical tools to enhance our understanding of lipid biology.

Abstract The subcellular distribution of lysosomes, the main degradative organelles of mammalian cells, responds to metabolic cues in a highly dynamic way. While lysosomal positioning due to amino acid levels is well-characterized, cholesterol-dependent regulation of lysosomal motility is incompletely understood. We explored impaired lysosomal cholesterol export using a mass spectrometry-based multi-OMICs approach, identifying widespread reallocation of resources and signaling pathway modulation. We identified increased phosphorylation at LAMTOR1 serine 56 in response to cholesterol level perturbations. We demonstrate that this phosphorylation site is sufficient to disrupt Rag GTPases/SLC38A9 binding to the Ragulator complex, inhibiting canonical mTORC1 and facilitating binding of BORC, therefore promoting lysosomal retrograde movement. LAMTOR1 S56 phosphorylation responds exclusively to depletion of lysosomal limiting membrane cholesterol, is facilitated by mTOR, and presents a negative feedback loop for amino acid independent displacement of Ragulator bound Rag GTPases, limiting canonical mTORC1 activity. Mass spectrometry data are available via ProteomeXchange with identifier PXD073489. Highlights Perturbation of lysosomal cholesterol homeostasis results in adaptation of cellular protein and lipid biosynthesis LAMTOR1 is phosphorylated at serine 56 via mTORC1 LAMTOR1 S56 phosphorylation is lysosomal membrane cholesterol dependent LAMTOR1 S56 phosphorylation disrupts binding of Rag GTPases to the Ragulator complex LAMTOR1 S56 phosphorylation promotes binding of Ragulator to BORC, facilitating lysosomal retrograde transport

Gangliosides are structurally complex glycosphingolipids that regulate cellular signaling via interactions with extracellular binding partners and by influencing protein function within the membrane. Their comprehensive analysis remains analytically challenging, in part due to low endogenous abundance, extensive structural diversity, and the frequent occurrence of isomeric species arising from both glycan and ceramide moieties. Here, we present a hydrophilic interaction liquid chromatography (HILIC) nanoelectrospray ionization tandem mass spectrometry (nanoESI-MS/MS) method specifically designed to achieve isomer-resolved analysis and molecular species-level quantification of gangliosides. To improve characterization of low-abundance fatty acyl fragments, the workflow was extended by combining nanoESI with online fraction collection and subsequent direct infusion. The method demonstrated stable chromatographic performance, high sensitivity, and reproducible quantitative results across ten mouse tissues. Application of the workflow enabled quantification of 80 ganglioside molecular species belonging to 18 subclasses and revealed pronounced tissue-specific differences in subclass distribution and ceramide composition. Overall, this study establishes a robust and sensitive platform for comprehensive ganglioside analysis, providing new opportunities to investigate ganglioside diversity, regulation, and function in complex biological systems.

Gangliosides are structurally complex glycosphingolipids that regulate cellular signaling via interactions with extracellular binding partners and by influencing protein function within the membrane. Their comprehensive analysis remains analytically challenging, in part due to low endogenous abundance, extensive structural diversity, and the frequent occurrence of isomeric species arising from both glycan and ceramide moieties. Here, we present a hydrophilic interaction liquid chromatography (HILIC) nanoelectrospray ionization tandem mass spectrometry (nanoESI-MS/MS) method specifically designed to achieve isomer-resolved analysis and molecular species-level quantification of gangliosides. To improve characterization of low-abundance fatty acyl fragments, the workflow was extended by combining nanoESI with online fraction collection and subsequent direct infusion. The method demonstrated stable chromatographic performance, high sensitivity, and reproducible quantitative results across ten mouse tissues. Application of the workflow enabled quantification of 80 ganglioside molecular species belonging to 18 subclasses and revealed pronounced tissue-specific differences in subclass distribution and ceramide composition. Overall, this study establishes a robust and sensitive platform for comprehensive ganglioside analysis, providing new opportunities to investigate ganglioside diversity, regulation, and function in complex biological systems.

AIMS:Platelets play a major role in thrombo-inflammatory cardiovascular diseases such as myocardial infarction. Although platelet function is crucially determined by kinases, the impact of Casein Kinase 2α (CK2α) on platelet activation during arterial thrombosis and myocardial remodeling following ischemia and reperfusion (I/R) injury is not known. METHODS AND RESULTS:Using platelet-specific deletion of Csnk2a1 in mice, the evaluation of the CK2α-dependent platelet phosphoproteome revealed a diminished phosphorylation of the IP3 receptor type-1 in Csnk2a1-deficient mice. This finding was accompanied by attenuated IP3-induced Ca2+ mobilization, impaired integrin αIIbβ3 activation, abrogated platelet aggregation and secretion, as well as defective spreading on fibrinogen in response to collagen-related peptide. Accordingly, without affecting primary hemostasis, thrombotic vascular occlusion in vivo was diminished in Csnk2a1-deficient mice. When subjected to a myocardial I/R injury model, these mice displayed improved cardiac outcome when compared with wildtype mice. Raman spectromics, spatial metabolomics and molecular approaches revealed locally a CK2α-dependent release of chondroitin sulfate and transforming growth factor-β from platelets, which was associated with significantly reduced ventricular fibrosis and improved heart function in Csnk2a1-deficient mice. CONCLUSION:Altogether, our results disclose CK2α as pivotal player in platelet activation and pathogenesis of post-ischemic myocardial remodeling, including myocardial fibrosis and left ventricular impairment following myocardial ischemia.

Neutrophil extracellular trap formation (NETosis) affects a wide variety of clinically relevant human diseases. Although lipid remodeling is essential for neutrophil function and membrane rupture during NETosis, the neutrophil lipidome and its dynamics have not been characterized. Thus, we establish a quantitative lipidome of human neutrophils comprising 1048 species across nine orders of magnitude and map its remodeling during NETosis. NET formation caused profound alterations in the phosphatidylinositol, phosphatidic acid, diacylglycerol (DG), and lyso-glycerophospholipid levels. Calcium- and reactive oxygen species-dependent NETosis pathways displayed distinct lipidomic trajectories yet converged on the significance of phospholipid lipase networks. Pharmacological inhibition of this networks altered lipid composition and markedly impaired NETosis, while DG treatment revoked the effect. Together, our findings reveal lipid remodeling as a fundamental determinant of NETosis and identify interconnected and dependent phospholipid lipase networks with downstream DG-dependent signaling as a potential therapeutic target in NET-associated diseases.

Synaptosomes (Syn) and synaptic junctions (SJ) are key neuronal compartments that have been widely characterized in omics studies to understand neurotransmitter- and signal transduction-related events. While synapses are lipid-rich, multiomics approaches integrating lipids and proteins remain largely underexplored. Liquid-liquid extraction (LLE), commonly used in lipidomics, offers significant potential for multiomics analyses by enabling the extraction of diverse molecular classes from a single sample. However, its impact on protein and phosphoprotein analysis in membrane-enriched samples has not been thoroughly investigated or compared to one-phase extraction methods. In this study, we assessed SIMPLEX (Simultaneous Metabolite, Protein, Lipid Extraction), an LLE-based method, against conventional acetone protein precipitation for mass spectrometry-based protein identification. SIMPLEX proved superior for proteomics and phosphoproteomics of SJ, achieving a 42% enrichment in membrane proteins compared to acetone precipitation. It enriched not only transmembrane proteins but also S-palmitoylated proteins. Enriched phosphoproteins included those with beta-transducin repeats (WD40), Armadillo repeats (ARM), and various transmembrane domains, highlighting the SIMPLEX potential and enhanced performance for multiomics analyses.

Large, randomized trials testing omega-3 polyunsaturated fatty acid (ω-3 PUFA) supplementation to reduce cardiovascular events have reported contradictory results. Interpretation of these trials is challenging, because different dosages and formulations of ω-3 PUFA were tested. Furthermore, the exact mechanisms for the reduction in cardiovascular events are unclear. In this study, we investigated the effects of ω-3 PUFA on platelet adhesion, degranulation, and aggregation in vitro and in patients with cardiovascular disease using different formulations of ω-3 PUFA. We also investigated the effects of ω-3 PUFA in rodent models of arterial thrombosis and in tail bleeding assays, including in cyclooxygenase-1 (COX-1)–deficient animals. The ω-3 PUFA eicosapentaenoic acid (EPA) dose-dependently reduced platelet adhesion, degranulation, and aggregation in vitro. Moreover, arterial thrombus formation in wild-type mice was inhibited by oral EPA administration before thrombus formation. Photoaffinity labeling and in silico docking analyses suggested a direct, competitive interaction of EPA and arachidonic acid at the level of COX-1. The COX-1 dependency of EPA’s inhibitory effects was confirmed by platelet-specific COX-1–deficient animals that had no reduction of thrombus burden by EPA. In patients with cardiovascular disease, switching from 2 grams of EPA twice daily to 1 gram of docosahexaenoic acid (DHA) (460 milligrams of EPA and 380 milligrams of DHA) once daily completely blunted the platelet inhibition achieved by EPA. Our results may partially explain contradictory results with different ω-3 PUFA formulations in clinical trials.

The scientific field of lipidomics has shown a constantly growing publication number in recent years, which is accompanied by an increasing need for quality standards. While the official shorthand nomenclature of lipids is a first and important step toward a reporting quality tool, an additional point score would reflect the quality of reported data at an even more detailed granularity. Thus, we propose a lipidomics scoring scheme that considers all the different layers of analytical information to be obtained by mass spectrometry, chromatography, and ion mobility spectrometry and awards scoring points for each of them. Furthermore, the scoring scheme is integrated with the annotation levels as proposed by the official shorthand nomenclature, with a point score, which roughly correlates with the annotated compound details. The merit of such a scoring system is the fact that it abstracts evidence for structural information into a number, which gives even the nonlipidomics expert an idea about the reporting, and by extension, data quality at first glance. Additionally, it could serve as an aid for internal quality control and for data quality assessment in the peer review process.

Phosphoinositides are a group of interconvertible lipids that are located in the membrane of eukaryotic cells. They turnover via complex network of reactions (called the phosphoinositide pathway) that respond rapidly to regulate many aspects of a cell's response to their environment. Given their low-abundance they are difficult to characterise experimentally. Here we utilise a new experimental method to generate an unusually large dataset that characterises the time-dependent changes in five membrane bound phospoinositides and a soluble inositide in platelet, downstream of its GPVI receptor, where we know the phosphoinositide pathway is particularly active. To shed light on regulatotory steps that are often opaque to experimentation we use this data within a mathematical and computational framework. We construct and assess eleven mathematical models that represent competing interpretations of the dominant mechanisms that regulate the pathway. We find that while four of the models can generate the available data only one model, that incorporates an additional pool of PtdIns, is consistent with the data and is able to successfully predict the effects of an inhibitor. We publish all models openly in a form that is easily usable and adaptable for other researchers to use alongside our or their own data. We studied how changes in the shape and magnitude of events that stimulate the phosphoinositide pathway affect its dynamics. Despite these perturbations, the abundance of Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) remained stable, consistent with findings reported in the literature.

In patients with a mild to moderate bleeding disorder (MBD) and abnormal light transmission aggregometry (LTA), a platelet function defect (PFD) is suspected. However, in many patients with PFD, the underlying mechanism remains elusive. Given the essential role of lipids in platelet signaling, platelet lipid profiles in MBD patients with unexplained PFD may provide valuable diagnostic and mechanistic insights. This prospective cohort study investigated platelet lipidomes in patients with PFD of unknown cause from the Vienna Bleeding Biobank (VIBB). Using a standardized lipidomics workflow, we analyzed platelets from 27 patients and 19 age- and sex-matched controls and found that sex-specific lipid shifts emerged exclusively within the patient cohort, with greater deviations in females. Furthermore, lipid alterations correlated with impaired platelet aggregation and were predictive of responses to ADP and TRAP-6 stimuli in LTA experiments. Baseline and stimulated platelet analyses in a female subgroup showed intrinsic lipidomic changes, including upregulated polyunsaturated triacylglycerols (PUFA-TG), acylcarnitines (CAR), and reduced lysophosphatidylethanolamines (LPE). This study emphasizes lipidomic profiling as a promising diagnostic tool for unexplained platelet dysfunction and highlights TG, CAR, and LPE as potential therapeutic targets. Further research into lipid-driven platelet regulation may advance personalized treatments and improve clinical outcomes for patients with MBD.

The brain is an exceptionally lipid-rich organ with a very complex lipid composition. Lipids are central in several neuronal processes, including membrane formation and fusion, myelin packing, and lipid-mediated signal transmission. Lipid diversity is associated with the evolution of higher cognitive abilities in primates, is affected by neuronal activity, and is instrumental for synaptic plasticity, illustrating that lipids are not static components of synaptic membranes. Several lines of evidence suggest that the lipid composition of synapses is unique and distinct from other neuronal subcompartments. Here, we delve into the nascent field of synaptoneurolipidomics, offering an overview of current knowledge on the lipid composition of synaptic junctions and technological advances that will allow us to study the impact on synaptic function.

Cholesterol serves as a biomarker in clinical- and life-sciences. The determination of abnormal levels can indicate several types of human diseases. However, the low polarity of free cholesterol makes it hardly accessible by (nano) electrospray ionization mass spectrometry (nESI-MS). As novel approach, the flexible microtube plasma (FμTP) for post-ionization allows the determination of low-polar compounds like cholesterol in combination with nESI-MS. Focusing on the analytical performance, the activated post-ionization leads to an increased cholesterol signal by a factor of 22. The repeatability and long-term stability could be successful evaluated by using a complex liver extract. Via the method of standard addition, a linear dynamic range of 1.7 orders of magnitude, a minimum detectability of 3.71 mg/L and a high accuracy (deviation: − 8.11 %) is demonstrated proofing the FμTP-nESI-MS as an excellent approach for a derivatization-free determination of cholesterol without the necessity of high-resolution Orbitrap devices or enhanced MS acquisition-methods.

The pronounced skin tropism and pan-antifungal resistance of Candida auris pose a serious global health threat. A key question in C. auris biology is how clinical isolates acquire amphotericin B resistance. Here we demonstrate that a carbonic sensing pathway (CSP) contributes to amphotericin B resistance by modulating mitochondrial energy functions in clinical C. auris isolates. Integrated transcriptomics and proteomics identify the carbonic anhydrase Nce103 and its transcription factors Rca1 and Efg1 as important regulatory components of the CSP. The conversion of CO2 into bicarbonate sustains energy metabolism required for colonization and fitness on human skin and in nutrient-limited microenvironments. We also show that bacterial skin colonizers engage urease to release CO2 that sustains C. auris fitness and skin colonization. These findings highlight therapeutic options to re-sensitize C. auris to antifungal treatments, as well as to prevent skin colonization by blocking the CSP.

Several oxylipins are potent lipid mediators that regulate diverse aspects of health and disease and whose quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) presents substantial technical challenges. As members of the lipidomics community, we developed technical recommendations to ensure best practices when quantifying oxylipins by LC-MS.

An animal's metabolic state strongly influences its behavior. Hungry animals prioritize food-seeking and feeding behaviors, while sated animals suppress these behaviors to engage in other activities. Additionally, neuronal activity and synaptic transmission are among the most energy-expensive processes. However, neurons do not uptake nutrients from the circulation. Instead, glia fulfill this highly evolutionarily conserved function in addition to modulating neuronal activity and behavior. However, how different glia subtypes sense metabolic state and modulate behavior is incompletely understood. Here, we unravel two types of glia-mediated modulation of metabolic-state-dependent behavior. In food-deprived flies, astrocyte-like and perineurial glia promote foraging and feeding, respectively, while cortex glia suppress these behaviors. We further show that adenosine and adenosine receptors modulate intracellular calcium levels in these glia subtypes, which ultimately controls behavior. This study reveals a mechanism of how different glia subtypes sense an animal's metabolic state and modulate its behavior accordingly.

Signaling lipids are key players in cellular processes. Despite their importance, no method currently allows their comprehensive monitoring in one analytical run. Challenges include a wide dynamic range, isomeric and isobaric species, and unwanted interaction along the separation path. Herein, we present a sensitive and robust targeted liquid chromatography-mass spectrometry (LC-MS/MS) approach to overcome these challenges, covering a broad panel of 17 different signaling lipid classes. It involves a simple one-phase sample extraction and lipid analysis using bioinert reversed-phase liquid chromatography coupled to targeted mass spectrometry. The workflow shows excellent sensitivity and repeatability in different biological matrices, enabling the sensitive and robust monitoring of 388 lipids in a single run of only 20 min. To benchmark our workflow, we characterized the human plasma signaling lipidome, quantifying 307 endogenous molecular lipid species. Furthermore, we investigated the signaling lipidome during platelet activation, identifying numerous regulations along important lipid signaling pathways. This highlights the potential of the presented method to investigate signaling lipids in complex biological systems, enabling unprecedentedly comprehensive analysis and direct insight into signaling pathways.

BACKGROUND:Aortic valve disease (AVD) is associated with high mortality and morbidity. To date, there is no pharmacological therapy available to prevent AVD progression. Because valve calcification is the hallmark of AVD and S1P (sphingosine-1-phosphate) plays an important role in osteogenic signaling, we examined the role of S1P signaling in aortic stenosis disease. METHODS:AVD progression and its consequences for cardiac function were examined in a murine wire injury-induced AVD model with and without pharmacological and genetic modulation of S1P production, degradation, and receptor signaling. S1P was measured by liquid chromatography-mass spectrometry. Calcification of human valvular interstitial cells and their response to biomechanical stress were analyzed in the context of S1P signaling. Human explanted aortic valves from patients undergoing aortic valve replacement and cardiovascular magnetic resonance imaging were analyzed for S1P by liquid chromatography-mass spectrometry. RESULTS:Raising S1P concentrations in mice with injury-induced AVD by pharmacological inhibition of its sole degrading enzyme S1P lyase vastly enhanced AVD progression and impaired cardiac function resembling human disease. In contrast, low S1P levels caused by SphK1 (sphingosine kinase 1) deficiency potently attenuated AVD progression. We found S1P/S1PR2 (S1P receptor 2) signaling to be responsible for the adverse S1P effect because S1PR2-deficient mice were protected against AVD progression and its deterioration by high S1P. It is important to note that pharmacological S1PR2 inhibition administered after wire injury successfully prevented AVD development. Mechanistically, biomechanical stretch stimulated S1P production by SphK1 in human valvular interstitial cells as measured by C17-S1P generation, whereas S1P/S1PR2 signaling induced their osteoblastic differentiation and calcification through osteogenic RUNX2/OPG signaling and the GSK3β-Wnt-β-catenin pathway. In patients with AVD, stenotic valves exposed to high wall shear stress had higher S1P content and increased SphK1 expression. CONCLUSIONS:Increased systemic or local S1P levels lead to increased valvular calcification. S1PR2 antagonists and SphK1 inhibitors may offer feasible pharmacological approaches to human AVD in prophylactic, disease-modifying or relapse-preventing manners.