Data acquisition consisted of alternating FTMS and FTMS/MS scans with class-specific acquisition settings. Lipids were identified using LipidXplorer and MFQL files.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Direct infusion MS - positive","Direct infusion MS - negative"],"direct_infusion_protocol":["Crude lipid extracts were infuzed into Thermo Scientific Q-exactive using TriVersa NanoMate
"],"publication":["VPS13C/PARK23 initiates lipid transfer and membrane remodeling for efficient lysosomal repair. 10.1038/s41467-026-75145-y. PMID:42393076"],"submitter_affiliation":["Danish Cancer Institute"],"submitter_name":["Kenji Maeda"],"organism_part":["Lysosome","HeLa cell","PNF","solvent"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["For lipidomics samples, cells were washed once with PBS and then incubated for 12 min at 37 °C in Opti-MEM in the presence or absence of 1 mM LLOMe. After incubation, the cells were washed twice with ice-cold PBS, detached from the plates by scraping with a rubber policeman in 16 mL ice-cold PBS, transferred to a 50 mL Falcon tube on ice, and centrifuged at 500 x g for 5 min at 4 °C. All steps from here were performed on ice or at 4 °C. Cell pellets were gently resuspended in 10 mL ice-cold MIB buffer (10 mM HEPES, 0.21 M mannitol, 0.070 M sucrose, pH 7.5) and centrifuged at 1000 x g for 5 min. This step was repeated once. Next, the cell pellets were resuspended in 0.8 mL freshly prepared and filtered MIB4 buffer (MIB buffer supplemented with 0.5 mM DTT, 0.5% fatty acid-free BSA (Sigma Aldrich), 25 units/mL Benzonase (Sigma Aldrich) and 1× cOmplete Mini, EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics)), taken up into a 1 mL Luer-Lok syringe (BD) and passed 25 times through a Balch homogenizer with a tungsten carbide ball of 8.012 mm. The cell lysates were centrifuged at 1500 x g for 10 min. The supernatants were transferred to fresh tubes and centrifuged again at 1500 x g for 15 min to generate a post-nuclear supernatant. For affinity purification of lysosomes, anti-GFP IgG microbeads (μMACS GFP Isolation Kit, Miltenyi Biotec, 130-091-125) were washed in MIB4 buffer, and 90 μL of a 1:1 bead:buffer slurry was mixed with 500 μL of postnuclear supernatant. After 1 h of gently mixing on a rotor at an angle of 45°, the suspensions were loaded onto MS columns (Miltenyi Biotec, 130-042-201) that were pre-equilibrated in MIB4 buffer and mounted on a magnetic stand (Miltenyi Biotec, 130-042-109). For the preparation of lipidomics samples, the columns were washed once with 500 μL MIB4 buffer, three times with 500 μL MIB2 buffer (MIB4 buffer lacking Benzonase and protease inhibitors), and eluted with 600 μL MIB buffer. The eluates were equally divided for immunoblot and proteomics analysis. Microbeads were washed once in 200 μL MIB+ buffer (MIB2 buffer without BSA) and collected by centrifugation at 21,100 x g for 20 min.
Lysosomal lipidomics
Microbead pellets with bound lysosomes and corresponding whole cells were subjected to lipid extraction as previously described[1]. Briefly, microbeads were resuspended in 200 μL 155 mM ammonium bicarbonate, mixed with 12.5 μL internal lipid standard mix (provided as Supplementary Data 2) and 988 μL chloroform:methanol 2:1 (v/v), and then shaken in a thermomixer at 2000 rpm at 4 °C for 15 min. After centrifugation (2000 × g, 2 min, 4 °C), the lower phase containing lipids was transferred to new tubes and dried in a vacuum centrifuge for 75 min. The dried lipids were resuspended in 100 μL chloroform:methanol 1:2 (v/v) and subjected to shotgun lipidomics as previously described.
Ref
[1] Nielsen, I.Ø., Vidas Olsen, A., Dicroce-Giacobini, J., Papaleo, E., Andersen, K.K., Jaattela, M., Maeda, K. and Bilgin, M., 2020. Comprehensive evaluation of a quantitative shotgun lipidomics platform for mammalian sample analysis on a high-resolution mass spectrometer. Journal of the american society for mass spectrometry, 31(4), pp.894-907.
"],"organism":["blank","Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS14737"],"author":["Kenji Maeda. Danish Cancer Institute. Strandboulevarden 49, Copenhagen, Denmark. kenjim@cancer.dk."],"data_transformation_protocol":["The ion intensities and mz were extracted from raw data files using lipidXplorer (and class-specific MFQL files), which also assiged them to lipid species.
"],"study_factor":["Treatment","Replicate"],"submitter_email":["kenjim@cancer.dk"],"sample_collection_protocol":["HeLa cells in culture were treated with LLOMe (or ctrl). After harvesting, the cells were lysed, and lysosomes were IPed from the post-nuclear fraction (PNF). The whole-cells, PNF, and lysosomes were then submitted to shotgun lipidomics.
"],"omics_type":["Metabolomics"],"study_design":["triversa nanomate","Homo sapiens","PNF","Lipidomics","negative ionization mode","LipidXplorer","blank/buffer","semi-targeted analysis","Lipidomics Core Facility at the Danish Cancer Institute","Thermo Scientific Q Exactive","positive ionization mode","experimental blank","Cell","lysosome"],"curator_keywords":["triversa nanomate","Homo sapiens","PNF","Lipidomics","negative ionization mode","LipidXplorer","blank/buffer","semi-targeted analysis","Lipidomics Core Facility at the Danish Cancer Institute","Thermo Scientific Q Exactive","positive ionization mode","experimental blank","Cell","lysosome"],"mass_spectrometry_protocol":["Lipids in crude extracts were measured in the positive and negative ionization modes, using a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) . Each ionization mode was further subdivided with varying mz ranges and between MS and MS/MS. Lipid class-specific acquisition settings and fragmentation strategies were as previously described[1].
Ref
[1] Nielsen, I.Ø., Vidas Olsen, A., Dicroce-Giacobini, J., Papaleo, E., Andersen, K.K., Jaattela, M., Maeda, K. and Bilgin, M., 2020. Comprehensive evaluation of a quantitative shotgun lipidomics platform for mammalian sample analysis on a high-resolution mass spectrometer. Journal of the american society for mass spectrometry, 31(4), pp.894-907.
"],"additional_accession":[]},"is_claimable":false,"name":"Shotgun lipidomics of HeLa whole-cell lysates and lysosomes following LLOMe treatment","description":"Perturbations in lysosome integrity are tightly linked to neurological disorders and ageing, but the underlying pathogenic mechanisms are incompletely understood. Using an unbiased proteomic approach, we here identified the bridge-like lipid transport protein VPS13C/PARK23 as a key component of a global early response pathway to lysosome damage. VPS13C readily binds lysosomes under mechanical or osmotic tension in anticipation of membrane lesions. The latter trigger a conformational change in the protein’s C-terminus, involving its ATG2C domain acting as sensor of damage-induced lipid packing defects. We show that ER-lysosome contacts formed by VPS13C provide critical binding platforms for OSBP/ORPs to enable efficient ER wrapping of damaged lysosomes. A chemical approach to assess directional ER-to-lysosome lipid transport revealed that VPS13C is essential for large-scale lipid delivery to acutely damaged lysosomes to facilitate their repair. Our findings offer new mechanistic insights into how loss-of-function mutations in VPS13C may enhance the risk of Parkinson’s disease.
This study contains shotgun lipidomics data from HeLa cells treated with L-leucyl-L-leucine methyl ester (LLOMe), a lysosomotropic agent that induces lysosomal membrane damage. Lipid profiles were obtained from both whole-cell lysates and isolated lysosomal fractions. Lipids were extracted and quantified by mass spectrometry-based shotgun lipidomics.
","dates":{"publication":"2026-07-03","submission":"2026-06-10"},"accession":"MTBLS14737","cross_references":{"pubmed":["42393076"]}}