Lipidome Unsaturation Affects the Morphology and Proteome of the Drosophila Eye.

Organisms respond to dietary and environmental challenges by altering the molecular composition of their glycerolipids and glycerophospholipids (GPLs), which may favorably adjust the physicochemical properties of lipid membranes. However, how lipidome changes affect the membrane proteome and, eventually, the physiology of specific organs is an open question. We addressed this issue in Drosophila melanogaster, which is not able to synthesize sterols and polyunsaturated fatty acids but can acquire them from food. We developed a series of semisynthetic foods to manipulate the length and unsaturation of fatty acid moieties in GPLs and singled out proteins whose abundance is specifically affected by membrane lipid unsaturation in the Drosophila eye. Unexpectedly, we identified a group of proteins that have muscle-related functions and increased their abundances under unsaturated eye lipidome conditions. In contrast, the abundance of two stress response proteins, Turandot A and Smg5, is decreased by lipid unsaturation. Our findings could guide the genetic dissection of homeostatic mechanisms that maintain visual function when the eye is exposed to environmental and dietary challenges.

Critical shifts in lipid metabolism promote megakaryocyte differentiation and proplatelet formation.

During megakaryopoiesis, megakaryocytes (MK) undergo cellular morphological changes with strong modification of membrane composition and lipid signaling. Here we adopt a lipid-centric multiomics approach to create a quantitative map of the MK lipidome during maturation and proplatelet formation. Data reveal that MK differentiation is driven by an increased fatty acyl import and de novo lipid synthesis, resulting in an anionic membrane phenotype. Pharmacological perturbation of fatty acid import and phospholipid synthesis blocked membrane remodeling and directly reduced MK polyploidization and proplatelet formation resulting in thrombocytopenia. The anionic lipid shift during megakaryopoiesis was paralleled by lipid-dependent relocalization of the scaffold protein CKIP-1 and recruitment of the kinase CK2α to the plasma membrane, which seems to be essential for sufficient platelet biogenesis. Overall, this study provides a framework to understand how the MK lipidome is altered during maturation and the impact of MK membrane lipid remodeling on MK kinase signaling involved in thrombopoiesis.

Eye proteome of Drosophila melanogaster.

Drosophila melanogaster is a popular model organism to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies, aging, light-induced damage, or dietary deficiencies. Large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which led to the discovery of key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins, including the absolute (molar) quantities of 43 proteins in the eye of adult male Drosophila reared on standard laboratory food. This work provides a generic and expandable resource for further genetic, pharmacological, and dietary studies.

Accurate Sphingolipid Quantification Reducing Fragmentation Bias by Nonlinear Models.

Quantitative sphingolipid analysis is crucial for understanding the roles of these bioactive molecules in various physiological and pathological contexts. Molecular sphingolipid species are typically quantified using sphingoid base-derived fragments relative to a class-specific internal standard. However, the commonly employed "one standard per class" strategy fails to account for fragmentation differences presented by the structural diversity of sphingolipids. To address this limitation, we developed a novel approach for quantitative sphingolipid analysis. This approach utilizes fragmentation models to correct for structural differences and thus overcomes the limitations associated with using a limited number of standards for quantification. Importantly, our method is independent of the internal standard, instrumental setup, and collision energy. Furthermore, we integrated this method into a user-friendly KNIME workflow. The validation results illustrate the effectiveness of our approach in accurately quantifying ceramide subclasses from various biological matrices. This breakthrough opens up new avenues for exploring sphingolipid metabolism and gaining insights into its implications.

LipidSpace: Simple Exploration, Reanalysis, and Quality Control of Large-Scale Lipidomics Studies.

Lipid analysis gained significant importance due to the enormous range of lipid functions, e.g., energy storage, signaling, or structural components. Whole lipidomes can be quantitatively studied in-depth thanks to recent analytical advancements. However, the systematic comparison of thousands of distinct lipidomes remains challenging. We introduce LipidSpace, a standalone tool for analyzing lipidomes by assessing their structural and quantitative differences. A graph-based comparison of lipid structures is the basis for calculating structural space models and subsequently computing lipidome similarities. When adding study variables such as body weight or health condition, LipidSpace can determine lipid subsets across all lipidomes that describe these study variables well by utilizing machine-learning approaches. The user-friendly GUI offers four built-in tutorials and interactive visual interfaces with pdf export. Many supported data formats allow an efficient (re)analysis of data sets from different sources. An integrated interactive workflow guides the user through the quality control steps. We used this suite to reanalyze and combine already published data sets (e.g., one with about 2500 samples and 576 lipids in one run) and made additional discoveries to the published conclusions with the potential to fill gaps in the current lipid biology understanding. LipidSpace is available for Windows or Linux (https://lifs-tools.org).

Lipidome unsaturation affects the morphology and proteome of the Drosophila eye.

While the proteome of an organism is largely determined by the genome, the lipidome is shaped by a poorly understood interplay of environmental factors and metabolic processes. To gain insights into the underlying mechanisms, we analyzed the impacts of dietary lipid manipulations on the ocular proteome of Drosophila melanogaster . We manipulated the lipidome with synthetic food media that differed in the supplementation of an equal amount of saturated or polyunsaturated triacylglycerols. This allowed us to generate flies whose eyes had a highly contrasting length and unsaturation of glycerophospholipids, the major lipid class of biological membranes, while the abundance of other membrane lipid classes remained unchanged. By bioinformatically comparing the resulting ocular proteomic trends and contrasting them with the impacts of vitamin A deficiency, we identified ocular proteins whose abundances are differentially affected by lipid saturation and unsaturation. For instance, we unexpectedly identified a group of proteins that have muscle-related functions and increase their abundances in the eye upon lipidome unsaturation but are unaffected by lipidome saturation. Moreover, we identified two differentially lipid-responsive proteins involved in stress responses, Turandot A and Smg5, whose abundances decrease with lipid unsaturation. Lastly, we discovered that the ocular lipid class composition is robust to dietary changes and propose that this may be a general homeostatic feature of the organization of eukaryotic tissues, while the length and unsaturation of fatty acid moieties is more variable to compensate environmental challenges. We anticipate that these insights into the molecular responses of the Drosophila eye proteome to specific lipid manipulations will guide the genetic dissection of the mechanisms that maintain visual function when the eye is exposed to dietary challenges.

Flexible Microtube Plasma for the Consecutive-Ionization of Cholesterol in Nano-Electrospray Mass Spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is an established method for the identification of biomarkers. By nano-ESI (nESI), the polar molecular fraction of complex biological samples can be successfully ionized. In contrast, the less-polar free cholesterol, which serves as an important biomarker for several human diseases, is barely accessible by nESI. Although, complex scan functions of modern high-resolution MS devices are able to increase the signal-to-noise ratio, they are limited by the ionization efficiency of the nESI. One possible method to increase the ionization efficiency is the derivatization with acetyl chloride, however interferences with cholesteryl esters must be considered, so chromatographic separation or complex scan functions may be required. A novel approach to increase the yield of cholesterol ions of the nESI could be the application of a second consecutive-ionization process. This publication presents the flexible microtube plasma (FµTP) as a consecutive-ionization source, which allows the determination of cholesterol in nESI-MS analysis. Focusing on the analytical performance, the nESI-FµTP approach increases the cholesterol signal yield in a complex liver extract by a factor of 49. The repeatability and long-term stability could be successfully evaluated. A linear dynamic range of 1.7 orders of magnitude, a minimum detectability of 5.46 mg/L, and a high accuracy (deviation, -8.1%) demonstrates the nESI-FµTP-MS as an excellent approach for a derivatization-free determination of cholesterol.

Quantification of bulk lipid species in human platelets and their thrombin-induced release.

Lipids play a central role in platelet physiology. Changes in the lipidome have already been described for basal and activated platelets. However, quantitative lipidomic data of platelet activation, including the released complex lipids, are unavailable. Here we describe an easy-to-use protocol based on flow-injection mass spectrometry for the quantitative analysis of bulk lipid species in basal and activated human platelets and their lipid release after thrombin activation. We provide lipid species concentrations of 12 healthy human donors, including cholesteryl ester (CE), ceramide (Cer), free cholesterol (FC), hexosylceramide (HexCer), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM) and triglycerides (TG). The assay exhibited good technical repeatability (CVs < 5% for major lipid species in platelets). Except for CE and TG, the inter-donor variability of the majority of lipid species concentrations in platelets was < 30% CV. Balancing of concentrations revealed the generation of LPC and loss of TG. Changes in lipid species concentrations indicate phospholipase-mediated release of arachidonic acid mainly from PC, PI, and PE but not from PS. Thrombin induced lipid release was mainly composed of FC, PS, PC, LPC, CE, and TG. The similarity of the released lipidome with that of plasma implicates that lipid release may originate from the open-canalicular system (OCS). The repository of lipid species concentrations determined with this standardized platelet release assay contribute to elucidating the physiological role of platelet lipids and provide a basis for investigating the platelet lipidome in patients with hemorrhagic or thrombotic disorders.

High-throughput assessment identifying major platelet Ca2+ entry pathways via tyrosine kinase-linked and G protein-coupled receptors.

In platelets, elevated cytosolic Ca2+ is a crucial second messenger, involved in most functional responses, including shape change, secretion, aggregation and procoagulant activity. The platelet Ca2+ response consists of Ca2+ mobilization from endoplasmic reticulum stores, complemented with store-operated or receptor-operated Ca2+ entry pathways. Several channels can contribute to the Ca2+ entry, but their relative contribution is unclear upon stimulation of ITAM-linked receptors such as glycoprotein VI (GPVI) and G-protein coupled receptors such as the protease-activated receptors (PAR) for thrombin. We employed a 96-well plate high-throughput assay with Fura-2-loaded human platelets to perform parallel [Ca2+]i measurements in the presence of EGTA or CaCl2. Per agonist condition, this resulted in sets of EGTA, CaCl2 and Ca2+ entry ratio curves, defined by six parameters, reflecting different Ca2+ ion fluxes. We report that threshold stimulation of GPVI or PAR, with a variable contribution of secondary mediators, induces a maximal Ca2+ entry ratio of 3-7. Strikingly, in combination with Ca2+-ATPase inhibition by thapsigargin, the maximal Ca2+ entry ratio increased to 400 (GPVI) or 40 (PAR), pointing to a strong receptor-dependent enhancement of store-operated Ca2+ entry. By pharmacological blockage of specific Ca2+ channels in platelets, we found that, regardless of GPVI or PAR stimulation, the Ca2+ entry ratio was strongest affected by inhibition of ORAI1 (2-APB, Synta66) > Na+/Ca2+ exchange (NCE) > P2×1 (only initial). In contrast, inhibition of TRPC6, Piezo1/2 or STIM1 was without effect. Together, these data reveal ORAI1 and NCE as dominating Ca2+ carriers regulating GPVI- and PAR-induced Ca2+ entry in human platelets.

Ex vivo instability of lipids in whole blood: preanalytical recommendations for clinical lipidomics studies.

Reliability, robustness, and interlaboratory comparability of quantitative measurements is critical for clinical lipidomics studies. Lipids' different ex vivo stability in blood bears the risk of misinterpretation of data. Clear recommendations for the process of blood sample collection are required. We studied by UHPLC-high resolution mass spectrometry, as part of the "Preanalytics interest group" of the International Lipidomics Society, the stability of 417 lipid species in EDTA whole blood after exposure to either 4°C, 21°C, or 30°C at six different time points (0.5 h-24 h) to cover common daily routine conditions in clinical settings. In total, >800 samples were analyzed. 325 and 288 robust lipid species resisted 24 h exposure of EDTA whole blood to 21°C or 30°C, respectively. Most significant instabilities were detected for FA, LPE, and LPC. Based on our data, we recommend cooling whole blood at once and permanent. Plasma should be separated within 4 h, unless the focus is solely on robust lipids. Lists are provided to check the ex vivo (in)stability of distinct lipids and potential biomarkers of interest in whole blood. To conclude, our results contribute to the international efforts towards reliable and comparable clinical lipidomics data paving the way to the proper diagnostic application of distinct lipid patterns or lipid profiles in the future.

Niemann-Pick C1 protein regulates platelet membrane-associated calcium ion signaling in thrombo-occlusive diseases in mice.

BACKGROUND: Pathophysiologic platelet activation leads to thrombo-occlusive diseases such as myocardial infarction or ischemic stroke. Niemann-Pick C1 protein (NPC1) is involved in the regulation of lysosomal lipid trafficking and calcium ion (Ca2+) signaling, and its genetic mutation causes a lysosomal storage disorder. Lipids and Ca2+ are key players in the complex orchestration of platelet activation. OBJECTIVES: The present study aimed to determine the impact of NPC1 on Ca2+ mobilization during platelet activation in thrombo-occlusive diseases. METHODS: Using MK/platelet-specific knockout mice of Npc1 (Npc1Pf4∆/Pf4∆), ex vivo and in vitro approaches as well as in vivo models of thrombosis, we investigated the effect of Npc1 on platelet function and thrombus formation. RESULTS: We showed that Npc1Pf4∆/Pf4∆ platelets display increased sphingosine levels and a locally impaired membrane-associated and SERCA3-dependent Ca2+ mobilisation compared to platelets from wildtype littermates (Npc1lox/lox). Further, we observed decreased platelet. CONCLUSION: Our findings highlight that NPC1 regulates membrane-associated and SERCA3-dependent Ca2+ mobilization during platelet activation and that MK/platelet-specific ablation of Npc1 protects against experimental models of arterial thrombosis and myocardial or cerebral ischemia/reperfusion injury.

Eye proteome of Drosophila melanogaster.

The Drosophila melanogaster eye is a popular model to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies. For instance, the Drosophila eye has been used to investigate the impacts of ageing and environmental stresses such as light-induced damage or dietary deficiencies. Moreover, large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which includes key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins he adult Drosophila melanogaster eye and provide a generic and expandable resource for further genetic, pharmacological, and dietary studies.

Guiding the choice of informatics software and tools for lipidomics research applications.

Progress in mass spectrometry lipidomics has led to a rapid proliferation of studies across biology and biomedicine. These generate extremely large raw datasets requiring sophisticated solutions to support automated data processing. To address this, numerous software tools have been developed and tailored for specific tasks. However, for researchers, deciding which approach best suits their application relies on ad hoc testing, which is inefficient and time consuming. Here we first review the data processing pipeline, summarizing the scope of available tools. Next, to support researchers, LIPID MAPS provides an interactive online portal listing open-access tools with a graphical user interface. This guides users towards appropriate solutions within major areas in data processing, including (1) lipid-oriented databases, (2) mass spectrometry data repositories, (3) analysis of targeted lipidomics datasets, (4) lipid identification and (5) quantification from untargeted lipidomics datasets, (6) statistical analysis and visualization, and (7) data integration solutions. Detailed descriptions of functions and requirements are provided to guide customized data analysis workflows.

Vitamin A Deficiency Alters the Phototransduction Machinery and Distinct Non-Vision-Specific Pathways in the Drosophila Eye Proteome.

The requirement of vitamin A for the synthesis of the visual chromophore and the light-sensing pigments has been studied in vertebrate and invertebrate model organisms. To identify the molecular mechanisms that orchestrate the ocular response to vitamin A deprivation, we took advantage of the fact that Drosophila melanogaster predominantly requires vitamin A for vision, but not for development or survival. We analyzed the impacts of vitamin A deficiency on the morphology, the lipidome, and the proteome of the Drosophila eye. We found that chronic vitamin A deprivation damaged the light-sensing compartments and caused a dramatic loss of visual pigments, but also decreased the molar abundance of most phototransduction proteins that amplify and transduce the visual signal. Unexpectedly, vitamin A deficiency also decreased the abundances of specific subunits of mitochondrial TCA cycle and respiratory chain components but increased the levels of cuticle- and lens-related proteins. In contrast, we found no apparent effects of vitamin A deficiency on the ocular lipidome. In summary, chronic vitamin A deficiency decreases the levels of most components of the visual signaling pathway, but also affects molecular pathways that are not vision-specific and whose mechanistic connection to vitamin A remains to be elucidated.

A Current Encyclopedia of Bioinformatics Tools, Data Formats and Resources for Mass Spectrometry Lipidomics.

Mass spectrometry is a widely used technology to identify and quantify biomolecules such as lipids, metabolites and proteins necessary for biomedical research. In this study, we catalogued freely available software tools, libraries, databases, repositories and resources that support lipidomics data analysis and determined the scope of currently used analytical technologies. Because of the tremendous importance of data interoperability, we assessed the support of standardized data formats in mass spectrometric (MS)-based lipidomics workflows. We included tools in our comparison that support targeted as well as untargeted analysis using direct infusion/shotgun (DI-MS), liquid chromatography-mass spectrometry, ion mobility or MS imaging approaches on MS1 and potentially higher MS levels. As a result, we determined that the Human Proteome Organization-Proteomics Standards Initiative standard data formats, mzML and mzTab-M, are already supported by a substantial number of recent software tools. We further discuss how mzTab-M can serve as a bridge between data acquisition and lipid bioinformatics tools for interpretation, capturing their output and transmitting rich annotated data for downstream processing. However, we identified several challenges of currently available tools and standards. Potential areas for improvement were: adaptation of common nomenclature and standardized reporting to enable high throughput lipidomics and improve its data handling. Finally, we suggest specific areas where tools and repositories need to improve to become FAIRer.

Illustrated State-of-the-Art Capsules of the ISTH 2022 Congress.

The ISTH London 2022 Congress is the first held (mostly) face-to-face again since the COVID-19 pandemic took the world by surprise in 2020. For 2 years we met virtually, but this year's in-person format will allow the ever-so-important and quintessential creativity and networking to flow again. What a pleasure and joy to be able to see everyone! Importantly, all conference proceedings are also streamed (and available recorded) online for those unable to travel on this occasion. This ensures no one misses out. The 2022 scientific program highlights new developments in hemophilia and its treatment, acquired and other inherited bleeding disorders, thromboinflammation, platelets and coagulation, clot structure and composition, fibrinolysis, vascular biology, venous thromboembolism, women's health, arterial thrombosis, pediatrics, COVID-related thrombosis, vaccine-induced thrombocytopenia with thrombosis, and omics and diagnostics. These areas are elegantly reviewed in this Illustrated Review article. The Illustrated Review is a highlight of the ISTH Congress. The format lends itself very well to explaining the science, and the collection of beautiful graphical summaries of recent developments in the field are stunning and self-explanatory. This clever and effective way to communicate research is revolutionary and different from traditional formats. We hope you enjoy this article and will be inspired by its content to generate new research ideas.

Platelet lipid metabolism in vascular thrombo-inflammation.

The function of platelets - and thereby the balance between thrombosis and hemostasis - critically depends on their lipid composition. At the same time, platelets are capable of interacting with inflammatory cells by releasing lipids in a paracrine manner. Over the years, many studies have emphasized the importance of both, membrane and signaling lipids, in the signaling pathways underlying arterial thrombosis and chronic inflammation. In line with this, an imbalance of platelet lipid homeostasis is associated with thrombo-inflammatory diseases such as acute coronary syndrome. By establishing quantitative platelet lipidomic analysis, an opportunity has arisen to deepen our knowledge about platelet lipids regulating thrombo-inflammation and vice versa. Past and future investigations in this upcoming field are of great clinical importance since they will presumably pave the way for the identification of novel biomarkers. In addition, targeting specific regulators of the platelet lipid metabolism is a promising strategy to receive both anti-thrombotic and anti-inflammatory therapeutics and could be beneficial to a wide variety of patients with vascular thrombo-inflammatory diseases. This review summarizes the latest scientific findings in the field of platelet lipidomics research and does so by focusing on the metabolism of sphingolipids, oxylipins and phosphoinositides, which are affected by dynamic modifications in a pathophysiological manner. Further, this review elucidates the impact of these platelet lipids on thrombo-inflammatory cardiovascular diseases and highlights potential diagnostic and therapeutic targets.

Goslin 2.0 Implements the Recent Lipid Shorthand Nomenclature for MS-Derived Lipid Structures.

Goslin is the first grammar-based computational library for the recognition/parsing and normalization of lipid names following the hierarchical lipid shorthand nomenclature. The new version Goslin 2.0 implements the latest nomenclature and adds an additional grammar to recognize systematic IUPAC-IUB fatty acyl names as stored, e.g., in the LIPID MAPS database and is perfectly suited to update lipid names in LIPID MAPS or HMDB databases to the latest nomenclature. Goslin 2.0 is available as a standalone web application with a REST API as well as C++, C#, Java, Python 3, and R libraries. Importantly, it can be easily included in lipidomics tools and scripts providing direct access to translation functions. All implementations are open source.

Neddylation-dependent protein degradation is a nexus between synaptic insulin resistance, neuroinflammation and Alzheimer's disease.

BACKGROUND: The metabolic syndrome is a consequence of modern lifestyle that causes synaptic insulin resistance and cognitive deficits and that in interaction with a high amyloid load is an important risk factor for Alzheimer's disease. It has been proposed that neuroinflammation might be an intervening variable, but the underlying mechanisms are currently unknown. METHODS: We utilized primary neurons to induce synaptic insulin resistance as well as a mouse model of high-risk aging that includes a high amyloid load, neuroinflammation, and diet-induced obesity to test hypotheses on underlying mechanisms. RESULTS: We found that neddylation and subsequent activation of cullin-RING ligase complexes induced synaptic insulin resistance through ubiquitylation and degradation of the insulin-receptor substrate IRS1 that organizes synaptic insulin signaling. Accordingly, inhibition of neddylation preserved synaptic insulin signaling and rescued memory deficits in mice with a high amyloid load, which were fed with a 'western diet'. CONCLUSIONS: Collectively, the data suggest that neddylation and degradation of the insulin-receptor substrate is a nodal point that links high amyloid load, neuroinflammation, and synaptic insulin resistance to cognitive decline and impaired synaptic plasticity in high-risk aging.