Oxylipins are bioactive lipid mediators that play key roles in biological and pathological processes. Their remarkable chemical diversity makes their identification by untargeted LC-MS/MS analyses challenging. To date, effective solutions for their comprehensive characterization remain unavailable. Here, we present the first implementation of the recently refined Ion Identity Molecular Networking (IIMN) strategy to map the chemical space of oxylipins, together with a systematic evaluation of factors that hinder accurate annotation in MS/MS datasets. Building on recent mzmine software developments, we implemented a fully local strategy to perform the IIMN analysis without requiring web platforms or external tools. We established a high-quality MS/MS spectral library from 67 commercially available oxylipin standards using LC-MS data obtained in data-dependent acquisition mode. Integrating the detailed characterization of ion species generated during electrospray ionization into IIMN reduced network complexity. Across configurations, the modified cosine algorithm proved most effective for separating full-length from cyclized forms and for clustering oxylipins through structurally coherent relationships. Application of the IIMN workflow to mouse spleen extracts, in combination with our in-house and publicly available experimental MS/MS libraries, enabled the organization of oxylipins into molecular families, facilitating their structural characterization and the discovery of novel species. Although manual curation remained necessary for certain coeluting isomers and ambiguous fragments, the IIMN-based approach significantly improved network interpretability and understanding. Overall, this study establishes IIMN as a robust bioinformatic tool for decoding oxylipin diversity and provides a successful strategy for mapping their chemical space, characterizing them within samples, and discovering novel mediators in biological matrices. The new combined reference spectral library has been made publicly available and will serve as a valuable resource for future redox lipidomics research.

Plant oxylipins (PO), including phytoprostanes (PhytoPs) and phytofurans (PhytoFs), are bioactive markers of lipid oxidation derived from alpha-linolenic acid and are recognized as early indicators of food oxidation. This study evaluated PO formation during post-harvest processing of four Colombian cocoa bean clones (FSV 41, FEC 2, FEAR 5, and FMA 7) under two conditions: controlled fermentation with starter cultures and drying (T1), and natural fermentation and drying (T2), followed by storage. PhytoPs ranged from 1.237 to 236.072 ng g-1 and PhytoFs from 0.027 to 487.35 ng g-1. Ent-16-F1t-PhytoP and Ent-9-(RS)-12-epi-ST-Delta 10-13-PhytoF were the most representative compounds. Lower PO levels under T1 were observed in FEAR 5, FEC 2, and FMA 7 clones. Fermentation was identified as a key stage in PO generation, strongly influenced by genotype, time, and postharvest conditions. This work provides a comprehensive assessment of PO evolution during post-harvest and storage, improving understanding of fatty acid oxidation in cocoa and supporting food quality improvement.

Sacrolide A, a 14-membered macrolactone oxylipin, was first isolated from the edible freshwater cyanobacterium Aphanothece sacrum and identified as a potent antimicrobial and cytotoxic metabolite. Given the rising use of A. sacrum as a luxury food ingredient and concerns over potential toxicity from excessive consumption, we achieved the total synthesis of (+)-sacrolide A to enable further biological evaluation. Our highly convergent strategy involved the coupling of an alpha-hydroxylated aldehyde with a propargylic alkyne and macrolactonization as key steps, along with Wittig coupling and Noyori reduction, leading to an efficient 15-step longer linear sequence. This approach facilitated an in-depth assessment of sacrolide A's cytotoxicity on liver and colon cells, shedding light on its potential health implications and bioactive properties.

Stroke is a major pathology, but current diagnostic methods are prone to misclassification with other neurological conditions, e.g., epilepsy or migraine, which delays proper treatment and carries a poorer prognosis. Development of accurate biomarkers to diagnose stroke events could decrease misidentification occurrence and promote appropriate, timely management. Lipid peroxidation products are generated under oxidative stress conditions and have previously been shown to increase in stroke patients. As the reference standard for lipid peroxidation, oxylipins could be biomarkers for acute ischemic cerebrovascular syndrome (AICS) events. Moreover, the ability to triage at-risk patients could expedite the necessary procedures required to conclusively diagnose AICS incidents. A label-free, in vivo oxidative status (IVOS) technology measuring carbonylation was also tested to determine efficacy as a point-of-care assessment tool. Potential AICS patients were independently clinically classified as transient ischaemic attack (TIA) or minor ischaemic stroke (grouped as AICS), or mimics per our published FAST-IT Protocol, including radiological assessment. Plasma oxylipins were quantified using liquid chromatography tandem mass spectrometry while carbonylation was determined with IVOS technology. Oxylipin and IVOS metrics from diagnostic groups were compared using respective, univariate linear regressions. Concentrations of 4(RS)-4-F4t-NeuroP fragment (oxylipin) and Mean Fluorescence (ARBU) detected by mass spectrometry or IVOS technology, respectively, were higher in AICS patients compared with mimics. This is the first study to investigate an early point-of-care assessment device combined with a panel of oxylipins to identify markers of oxidative stress associated with transient or minor ischemic stroke incidents to improve diagnostic differentiation of AICS patients from mimics.

As lipidomics approaches its 25th anniversary, we explore how lipid research has matured over the years while highlighting emerging innovations that are expanding our ability to study these diverse, life-critical biomolecules. In particular, we showcase the community-driven, open-access databases, software, and educational resources made freely available through the ELIXIR Core Data Resource LIPID MAPS for the benefit of both established and new researchers.

BACKGROUND:Alzheimer's disease (AD) displays a sex imbalance; women represent two-thirds of cases and often progress faster. Lipid peroxidation contributes to AD neurotoxicity from the oxidation of fatty acids can. However, how sex modulates plasma lipid peroxidation across clinical stages remains poorly characterized. This study examines sex- and AD clinical stage-related patterns in these plasma compounds. METHODS:Plasma lipid peroxidation compounds were quantified by liquid chromatography/mass spectrometry in a clinical cohort stratified into 6 groups (cognitively unimpaired (without AD (n = 93), with AD (n = 27)), mild cognitive impairment (due to AD (n = 105), not due to AD (n = 45)), mild dementia (due to AD (n = 84), not due to AD (n = 34))). RESULTS:Across clinical groups, women showed a more severe clinical profile, while men showed higher neurofilament light chain (NfL) levels in several groups. Specifically, 10 lipid peroxidation compounds showed impaired levels in women with AD, while only 2 compounds in men. Notably, significant positive correlations were observed between certain lipid peroxidation compounds and cerebrospinal fluid (CSF) biomarkers exclusively in women. Positive correlations were observed between isoprostanes/neuroprostanes and some neuropyschological tests (CDR, MMSE, ADCS-ADL-MCI) in women, as well as mixed correlations between lipid peroxidation compounds and two tests (RBANS, FAQ) in men. Furthermore, multivariate analysis confirmed that clinical diagnosis was the main determining factor rather than biological sex, with 4 lipid peroxidation compounds (10-epi-10-F4t-NeuroP, 17-epi-17-F2t-dihomo-IsoP, total IsoP and total NeuroP) showing differences among clinical groups with distinct patterns of progression, specifically men showing higher levels in preclinical stages, while women showed more complex fluctuations throughout AD progression. CONCLUSIONS:Plasma lipid peroxidation follows sex-dependent biological patterns across clinical stages of AD, highlighting a wider variety of altered biomarkers in women, which underscores the need to evaluate sex-stratified approaches for the diagnosis and AD staging.

Staphylococcus aureus is a Gram-positive opportunistic pathogen and a top priority bacterium in the fight against antimicrobial resistance. Its high propensity to develop resistance, its high virulence, and its ability to form biofilms and persist intracellularly result in difficult-to-treat infections against, which new chemical classes are urgently needed. Here, we investigated the antibacterial activity of oxadiazolone-core derivatives (OX) against planktonic, intracellular, and biofilm-associated S. aureus. Among the tested compounds, MpPPOX exhibited a bactericidal effect on extracellular bacteria with an MIC similar to that of vancomycin; iBPOX mainly inhibited intracellular replication, while HPOX strongly impaired initial biofilm formation. These results prompted us to identify the potential target enzymes of the three OXs via activity-based protein profiling, combined with mass spectrometry. The antibiofilm HPOX compound was indeed found to primarily react with enzymes involved in biofilm formation and associated virulence, while iBPOX and the most active MpPPOX inhibitor targeted multiple (Ser/Cys)-based enzymes. Among these, the FabH protein has been confirmed as a vulnerable target of MpPPOX. Overall, this study underscores the multitarget nature of the OXs, which covalently bind to several (Ser/Cys)-based enzymes of interest. Such property makes them highly versatile chemotypes that could be used as broad-spectrum antimicrobial agents, notably by improving the antibiofilm activity of ineffective or poorly active drugs.

Oxidative and carbonyl stresses (COS), which damage brain cells through the accumulation of toxic reactive carbonyl species (RCS), are key players in the etiology of Alzheimer's disease (AD). Our group developed lipophenols, i.e. COS-targeting hybrid molecules combining polyunsaturated fatty acids (PUFAs) and alkyl-(poly)phenols. Among them, quercetin-3-O-docosahexaenoate-7-O-isopropyl (Quercetin-3-O-DHA-7-O-iPr or "Q-iP-DHA") afforded neuroprotection against acrolein-induced toxicity, reduced carbonyl stress, and lowered amyloid-beta secretion in neuroblastoma cells. To evaluate Q-iP-DHA in vivo, it was formulated into lipid nanocapsules (to allow solubilization) then administered intranasally to J20 transgenic mice, a model of AD. This approach was chosen to optimize blood-brain barrier (BBB) penetration. This delivery led to improvements in well-being, organizational skills and spatial memory. In addition, Q-iP-DHA treatment reduced hippocampal amyloid plaque numbers, normalized expression of the Receptor for Advanced Glycation End-products (RAGE), and decreased microglial activation, indicating anti-inflammatory effects. Overall, our preclinical findings suggest that intranasal administration of nanoformulated Q-iP-DHA may represent a promising multitarget therapeutic approach against AD.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are bioactive lipids with antidiabetic and anti-inflammatory effects, but the mechanisms responsible for their transport in blood are not well understood. In this study, we investigated potential serum carriers of FAHFAs. Microscale thermophoresis showed that binding of 9-PAHSA to serum albumin did not support albumin as the dominant carrier under the conditions tested. We therefore turned our attention to lipoproteins and extracellular vesicles, which were isolated from murine serum by density-based ultracentrifugation and characterized by proteomics and lipidomics. Lipidomic analysis revealed that chylomicrons, very-low-density lipoproteins (VLDL), and low-density lipoproteins (LDL) contain both FAHFAs and their storage form, triacylglycerol estolides (TG-EST). In contrast, no FAHFA-containing lipids were detected in serum extracellular vesicles. As a validation case study, we confirmed the presence of TG-EST in postprandial chylomicrons from human volunteers. Functional assays further showed that lipoprotein lipase (LPL), the enzyme that hydrolyzes triacylglycerols during lipoprotein metabolism, releases FAHFAs from TG-EST. LPL exhibited positional selectivity, releasing distinct PAHSA regioisomers with different efficiencies. Together, these results support the interpretation that FAHFA-related lipids are associated with chylomicron, VLDL, and LDL fractions in the postprandial setting and establish LPL-mediated lipolysis as a potential mechanism for the release and distribution of these bioactive lipids in the circulation.

F4-Neuroprostanes (F4-NeuroPs), oxidative metabolites of docosahexaenoic acid, act as bioactive lipid mediators enhancing sperm motility and induce capacitation-like changes in vitro. Their biological action is proposed to involve sperm ion channels, in particular ryanodine receptors (RyRs), which regulate intracellular calcium homeostasis. We evaluated the effects of dantrolene, a RyR inhibitor, on motility and vitality of a selected spermatozoa at different concentrations (10, 30, 50, 100 μM). Then sperm motility, acrosome integrity, and RyR localization following co-incubation with dantrolene (D50 or D100 μM) and 4-/10-F4t-NeuroPs (7 ng) were investigated. Acrosomal status was assessed using Pisum sativum agglutinin (PSA) staining and RyR localization by immunofluorescence. D50 was identified as the minimum effective dose to induce significant reductions in sperm motility. F4-NeuroPs significantly increased rapid progressive motility versus controls. Co-incubation with F4-NeuroPs + D50 reduced rapid motility and increased in situ and circular movement. The acrosome staining appeared altered or absent to different percentages, and RyR localization was also seen in the midpiece. These findings suggested that F4-NeuroPs enhance sperm motility via RyR-mediated pathways, as confirmed by dantrolene inhibition. Accordingly, our results underscore the physiological relevance of RyRs in sperm function and suggest new insights into lipid-based mechanisms regulating sperm motility.

Hybridalactone, agardhilactone, ecklonialactones, eiseniachlorides, and egregiachlorides, isolated from the marine macroalgae such as Laurencia hybrida, Agardhiella subulate, Ecklonia stolonifera, Eisenia bicyclis, and Egregia menziesii, are biosynthesized through a lipoxygenase (LOX)-mediated oxidative pathway involving polyunsaturated fatty acids (PUFAs). This enzymatic process generates hydroperoxide intermediates, which subsequently undergo regio- and stereospecific transformations to yield structurally diverse oxygenated metabolites, including plasmodiophorols and ectocarpins. These compounds are characterized by a conserved vinylcyclopentyl moiety, a structural hallmark critical to elucidating the mechanistic underpinnings of oxylipin biosynthesis in marine algae. The elucidation of these biosynthetic pathways enhances understanding of oxylipin enzymatic regulation and chemical diversity, while underscoring their ecological roles as signaling or defense molecules in marine ecosystems. Their unique structures and bioactivities offer potential for pharmacological and biotechnological applications, including novel bioactive agents. Investigating their production, structural complexity, and functionality is crucial for advancing marine natural product chemistry and exploring their ecological and industrial significance.

Oxylipins are bioactive lipid mediators derived from polyunsaturated fatty acids (PUFAs), with roles in oxidative stress responses, immunomodulation, and inflammation. While microalgae are recognized as valuable sources of oxylipins, their profiles remain less studied across different species and cultivation conditions. In this study, we characterized the non-enzymatic oxylipin profile of Chlorella sorokiniana grown under autotrophic and heterotrophic conditions to assess the influence of cultivation strategies on their production. A total of 22 isoprostanoids, mainly Phytoprostanes (PhytoP), Phytofurans (PhytoF), Isoprostanes (IsoP), and Neuroprostanes (NeuroP). Autotrophic cultivation resulted in a higher accumulation of isoprostanoids, particularly the alpha-linolenic acid (ALA) derivatives, PhytoP and PhytoF species, likely due to oxidative stress induced by fluctuating light and temperature conditions. In contrast, heterotrophic growth, performed under controlled conditions, yielded lower overall oxylipin levels highlighting the presence of 10(R)-10-F4t-NeuroP which was only present in heterotrophic Chlorella. We observed a correlation between the PUFA composition of Chlorella and its non-enzymatic oxylipin profile. Notably, several oxylipins identified in Chlorella have been associated with antiinflammatory, immunomodulatory, and neuroprotective properties, emphasizing the potential of this micro-alga as a source of high-value bioactive oxylipins. This study paves the way to the utilization of Chlorella as a source of bioactive oxylipins, as well as to develop cultivation strategies to enhance the production of these lipid mediators.

Quantitative analyses of per- and polyfluoroalkyl substances (PFAS) commonly rely on low-resolution targeted tandem mass spectrometry (MS/MS) due to its high sensitivity and relatively low operational threshold. Perfluoro-3,5,7,9-butaoxadecanoic acid (PFO4DA), an important constituent of the perfluoropolyether carboxylic acid (PFECA) family, has been widely detected in biotic and abiotic matrices. Nevertheless, marked interference has been observed in the MS/MS transition for PFO4DA (377 > 85) in biological samples, particularly in aquatic organisms, resulting in substantial overestimation of concentrations, reaching up to 66 ng/g. In this study, nontargeted molecular networking strategies, combined with the spectral simulation tools SIRIUS and QCxMS, were applied to identify the source of interference, which was confirmed as an oxygenated metabolite of docosahexaenoic acid (DHA) based on fragmentation patterns matching reference standards. The study further clarified that fatty acids generated the fragment ion [C4H5O2](-) (85.0296 Da), which overlapped with the [CF3O](-) (84.9907 Da) ion of PFECA compounds under low-resolution conditions, causing signal interference. Analysis of river water and fish samples revealed that this interference led to a 24-fold overestimation of PFO4DA bioaccumulation in aquatic organisms. These findings are essential for improving the selectivity of environmental exposure assessments for PFECA compounds.

BACKGROUND:Conventional sampling methods for clinical analysis make use of plasma or serum and require large blood volumes, cold storage, and specialized handling. This renders them impractical and invasive, especially when dealing with vulnerable groups of patients and the analysis of low-abundant and unstable compounds such as oxylipins. Dried blood micro-sampling methods are designed for collecting minimally invasive small-volume blood samples (<100 μL), providing advantages in stability, handling, and storage. The aim of this study is to develop and compare micro-sampling approaches that can be readily implemented in clinical analysis circumventing the barriers of conventional sampling methods. (96) RESULTS: This study assessed the performance of Pre-Cut Dried Blood Spots (PCDBS) and Volumetric Absorptive Micro-Sampling (VAMS®) devices for determining oxylipins, using small-volume liquid whole blood sampling as reference. A Liquid Chromatography - tandem Mass Spectrometry method was successfully developed and validated. Both PCDBS and VAMS® were prepared using 30 μL of spiked blood and dried for 2 h before extraction and analysis. Optimal results were achieved by pre-treating devices with antioxidant before sample deposition and avoiding the internal standard to undergo evaporation steps during preparation of micro-sampling devices. Long-term storage effects were assessed over a three-month period, and the micro-sampling devices were tested on umbilical cord blood samples from 35 newborns. For PCDBS and VAMS®, eight and nine oxylipins, respectively, showed comparable results to those obtained from liquid whole blood samples with mean concentrations ranging between 0.7 and 6.3 nM (140) SIGNIFICANCE: This study is the first to analyse oxylipins in umbilical cord blood using VAMS® and PCDBS micro-sampling devices, demonstrating their effectiveness for quantification at nM concentrations. It highlights the impact of sample collection, storage, and internal standard handling on lipid profiles. Stability is critical and cold storage (-20 °C) is mandatory. Future research focusing on the standardization of protocols for comparability among laboratories and enhancing sensitivity with improved LC-MS/MS techniques to expand accessible oxylipins are needed. (75).

Heart failure (HF) occurs when the heart fails to meet the body's demands. Differentiating and managing HF with Preserved Ejection Fraction (HFpEF) versus Reduced Ejection Fraction (HFrEF) remains challenging, as therapeutic strategies for HFpEF have largely been ineffective. Exercise intolerance is a hallmark of HFpEF, making the identification of biological pathways underlying exercise-related impairments particularly important. In this study, we integrated cardiopulmonary exercise testing with exercise stress echocardiography (CPET-ESE) and MS-based targeted lipid and epilipid profiling to investigate metabolic and immune dysregulation across different stages of HF. Due to the technical challenges and patient discomfort associated with venous blood collection during exercise, we employed a less invasive Dried Blood Spot (DBS) approach. For the first time, we successfully validated a method for targeted profiling of 52 oxylipins and 4 PUFAs in DBS samples, covering the entire inflammatory cascade. We established reliable DBS handling and storage procedures, with the addition of an internal standard mixture on filter paper ensuring high analyte recovery (93-107 %) and precision (RSD ≤12 %). Data from HF patients revealed significant differences in AA and anti-inflammatory omega-3 PUFA levels at rest. Furthermore, measuring AA and its epoxide metabolite, 8,9-EET, during exercise enabled clear differentiation between HFpEF, HFrEF, and stage A-B patients, potentially supporting earlier and more accurate diagnosis. Profiling alterations in free fatty acids and oxylipins could serve as a valuable tool for the in-depth pathophysiological characterization of HF patients.

This study investigates the self-assembly and host–guest complexation behaviour of novel resveratrol-based lipophenols (LipoResv)—resveratrol-4′-linoleate (Resv-4′-LA) and resveratrol-4′-docosahexaenoate (Resv-4′-DHA)—with hydroxypropyl-β-cyclodextrins (HP-β-CDs). These amphiphilic molecules display surfactant-like properties, forming micellar aggregates in aqueous media. Fluorescence spectroscopy was used to determine the critical micelle concentration (CMC), revealing that LipoResv exhibit significantly lower CMC values than their free fatty acids, indicating higher hydrophobicity. The formation of inclusion complexes with HP-β-CDs was evaluated based on changes in CMC values and further confirmed by dynamic light scattering (DLS) and molecular modelling analyses. Resv-4′-LA formed 1:1 complexes (Kc = 720 M−1), while Resv-4′-DHA demonstrated a 1:2 stoichiometry with lower affinity constants (K1 = 17 M−1, K2 = 0.18 M−1). Environmental parameters (pH, temperature, and ionic strength) significantly modulated CMC and binding constants. Computational docking and molecular dynamics simulations supported the experimental findings by revealing the key structural determinants of the host–guest affinity and micelle stabilization. Ligand efficiency (LE) analysis further aligned with the experimental data, favouring the unmodified fatty acids. These results highlight the versatile encapsulation capacity of HP-β-CDs for bioactive amphiphile molecules and support their potential applications in drug delivery and functional food systems.

This study investigates the evolution of lipids, oxylipins (phytoprostanes (PhytoPs) and phytofurans (PhytoFs)), and alkaloids in Mexican Coffea arabica leaves as coffee cherries mature. The impact of drying methods on leaf composition was also examined. Lipid extraction was performed using a binary MTBE/MeOH mixture. Fatty acid profiling was conducted by GC-FID. HPTLC was used to identify lipids (free fatty acids, acylglycerols, sterols, sterol esters, glycolipids, phospholipids, tocopherol), diterpenes (cafestol and kahweol), and alkaloids (caffeine and theobromine). A micro-HPLC-QTRAP analysis was used to identify and quantify oxylipins. The drying method influenced the leaves total lipid extraction. Freeze-dried leaves (HL) had higher lipid content than vacuum oven-dried leaves (HS). Total lipids decreased and oxylipin content varied with cherry maturity. The predominant fatty acid in coffee leaves was C18:3. This study reveals coffee leaves as a significant source of PhytoPs and PhytoFs for the first time.

To promote sustainable aquaculture, plant-based ingredients are increasingly being used to replace fish meal (FM) and fish oil (FO) in diets, affecting critical fatty acid (FA) profiles in broodstock diets. These profiles are essential for reproduction and offspring survival. The absence of traditional fish-derived nutrients results in an altered FA composition, particularly a reduction in omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs). This change is detrimental to trout reproduction over the long term. Current research gaps, especially regarding FA profiles and related metabolites, limit our understanding of the reproductive consequences of omega-3 deficient plant-based diets. To fill this knowledge gap, a two-year trial was conducted to compare the reproductive effects (spawning performances, egg quality, embryo development) of two diets on rainbow trout females: a plant-based diet containing linseed (10.8 %), palm (5 %), and sunflower oils (2.7 %), and a commercial diet with high FM (27 %) and FO (12.1 %) content. Both diets were similar in terms of protein (44 %), lipids (23.4 %), and energy (25 kJ/g). The study spanned from initial feeding to first reproduction, and fatty acid profiles and metabolites (selected enzymatic and non enzyamatic metabolites from FA) were examined in the broodstock diet, female muscle tissue, eggs, and fry. Trout fed a plant-based diet showed reduced reproductive traits such as lower weight and fecundity, but egg and embryo quality remained high. Survival and hatching rates were similar to those on commercial diets. The study revealed for the first time the critical role of maternal diet in the generation of lipid metabolites in the offspring, particularly DHA (F-4-neuroprostane, maresin, 14-HDoHE) and AA (F-2-isoprostane, prostaglandin E and F2, 8-12- 15-HETEs) oxylipins, highlightingthe complex dietary influences on fish reproduction. This work underlines that a plant-based diet significantly affects the the reproductive performance of broodstock trout by altering fatty acid profiles and metabolite levels and highlights the delicate balance between dietary components, oxylipins, and reproductive functions in fish, suggesting the need for more comprehensive studies of their interrelated effects.

Lipids play a critical role in the physiology, life cycle, and pathogenicity of mycobacteria. They largely participate in host-pathogen interactions and fulfill important functions ranging from cell wall biosynthesis/maintenance, bacterial growth dynamics, and long-term persistence. In that context, triacylglycerol, a specific subtype of neutral lipid, is stored as intrabacterial lipid inclusions (ILI), which have been described as important structures for long-term survival and persistence within the host. However, the enzymes involved in ILI formation and degradation remain largely undefined. Using bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP) of newly synthesized oxadiazolone (OX), cyclophostin, and cyclipostins (CyC) probes, we report the direct capture of target proteins in Mycobacterium abscessus growing under carbon excess and nitrogen-deprived in vitro conditions that promote triacylglycerol production and ILI formation. This approach led to the identification of a set of 65 enzymes potentially involved in global processes related to ILI anabolism. Among these, the long-chain-fatty-acid--CoA ligase MAB_1978c/FadD15 has been validated as a pivotal enzyme that colocalizes on ILI and as a major contributor in ILI formation in M. abscessus.