Data evaluation was done using LipidXplorer (1). LipidXplorer mfql queries were applied to detect precursor ions in MS scans and their corresponding fragment ions in MS/MS scans within the m/z range of 500-1000. Selection and quantification of internal standards and lipid species were done using an R-based in-house-developed software (ShinyLipidcountr_alpha4). The amount [pmol] for endogenous molecular lipid species was calculated based on the intensities of the corresponding internal standards and normalized to the sample volumes used for extraction to obtain lipid species concentrations [µM].
(1) Schuhmann, K.; Thomas, H.; Ackerman, J.M.; Nagornov, K.O.; Tsybin, Y.O.; Shevchenko, A., 'Intensity-Independent Noise Filtering in FT MS and FT MS/MS
Spectra for Shotgun Lipidomics', Analytical Chemistry, 2017, 89, 046−7052
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Direct infusion MS - positive","Direct infusion MS - negative"],"direct_infusion_protocol":["Samples were injected using a robotic electrospray infusion device Triversa Nanomate (Advion Biosciences) with chip-based ionization (HD-D ESI Chip, Advion Biosciences).
"],"publication":["Membrane lipid as cofactor of cargo uptake and release by box-like lipid transfer proteins."],"submitter_name":["Britta Brügger"],"submitter_affiliation":["Heidelberg University Biochemistry Center (BZH)"],"organism_part":["mixture","Liposome"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["Mass spectrometric quantitative untargeted lipid analysis of samples was performed as follows. Lipid extractions were performed in the presence of internal lipid standards using an acidic liquid-liquid extraction (ABD) method (1). Extraction was performed using 30 µL for supernatants, and 2 µL for liposomes. Lipid extractions were performed in the presence of internal lipid standards for each analyzed lipid class, with the standards resembling the structure of the endogenous lipid species. Following this approach, a relative quantification of lipid species was performed. Lipid standards were added prior to extractions, using a master mix consisting of 50 pmol phosphatidylcholine (PC, 18:1-d7/15:0, AvantiResearch), 40 pmol phosphatidylethanolamine (PE, 18:1-d7/15:0, AvantiResearch), 5 pmol ceramide (Cer, d18:1 with N-acylated 16:0-d9 and t18:0 with N-acylated 16:0-d9, AvantiResearch). The final chloroform phase was evaporated under a gentle stream of nitrogen at 37 °C. Samples were directly subjected to mass spectrometric analysis.
(1) Bligh, E.G.; Dyer, W.J., 'A Rapid Method for Total Lipid Extraction and Purification', Canadian Journal of Bio-Chemistry and Physiology, 1959, 37, 911-917
"],"organism":["reference compound; blank","reference compound; Escherichia coli","reference compound"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS15933"],"author":["Britta Brügger. Heidelberg University Biochemistry Center (BZH). INF 328, 69120 Heidelberg, Germany. britta.bruegger@bzh.uni-heidelberg.de."],"data_transformation_protocol":["Raw data was filtered and converted using PeakStrainer (1).
(1) Schuhmann, K.; Thomas, H.; Ackerman, J.M.; Nagornov, K.O.; Tsybin, Y.O.; Shevchenko, A., 'Intensity-Independent Noise Filtering in FT MS and FT MS/MS
Spectra for Shotgun Lipidomics', Analytical Chemistry, 2017, 89, 046−7052
"],"study_factor":["Experiment","Lipid transfer protein"],"submitter_email":["britta.bruegger@bzh.uni-heidelberg.de"],"sample_collection_protocol":["Heavy donor liposomes (Input LUV1 = POPC:POPE:Ceramide, 76:20:4 mol%; Input LUV2 = POPC:POPE 80:20 mol%; Input LUV3 = POPC:POPE:TAG, 76:20:4 mol%; all lipids from Avanti Research) were incubated in presence of buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 125 mM sucrose), 10 μM STARD11 wild-type (WT) or variant R478N (R478N). His-tagged proteins were recombinantly expressed in BL21(D3) cells and purified by NiNTA agarose followed by size exclusion chromatography. After pelleting liposomes incubated with buffer or the indicated proteins, supernatants (Sup) were gathered and analyzed by nanoESI-DI-MS/MS based lipidomics.
Liposome input samples and supernatants were aliquoted, frozen in liquid nitrogen and stored at -80°C.
"],"omics_type":["Metabolomics"],"study_design":["reference compound; Escherichia coli","Advion TriVersa NanoMate","Liposome","lipid homeostasis","Lipidomics","STARD","lipid transfer protein","reference compound mix","semi-targeted analysis","experimental sample","data-independent acquisition","reference compound; blank","Thermo Scientific Q Exactive","reference compound","mixture","parallel reaction monitoring","non-vesicular lipid transport","allostery"],"curator_keywords":["reference compound; Escherichia coli","Advion TriVersa NanoMate","Liposome","lipid homeostasis","Lipidomics","STARD","lipid transfer protein","reference compound mix","semi-targeted analysis","experimental sample","data-independent acquisition","reference compound; blank","reference compound","Thermo Scientific Q Exactive","mixture","parallel reaction monitoring","non-vesicular lipid transport","allostery"],"mass_spectrometry_protocol":["Lipid extracts were resuspended in 7.5 mM ammonium formate in 60 µl isopropanol:methanol:chloroform 4:2:1 (v/v/v) including 1.5 µM 12-{[(cyclohexylamino)carbonyl]amino}dodecanoic acid (CUDA) for lock mass reference compound. Samples were analyzed on a Q Exactive, coupled to a robotic electrospray infusion device Triversa Nanomate (Advion Biosciences) with chip-based ionization (HD-D ESI Chip, Advion Biosciences). Measurement preparation in 96well plate format (Eppendorf twin tec 96), 5 µl aliquots of the resuspended lipid extracts were diluted 1:3 in 15 µl 7.5 mM ammonium formate in isopropanol:methanol:chloroform (v:v:v, 4:2:1) including 1.5 µM CUDA for lock mass reference in 96-wells.
Mass spectrometric data were acquired in positive- and negative-ion modes using full-MS SIM, targeted SIM, and parallel reaction monitoring (PRM). In both ion modes, full-MS SIM spectra were acquired over m/z 320–1000 at a resolution of 140,000, with an AGC target of 3e6, a maximum injection time of 3000 ms, and one microscan per scan. Targeted SIM spectra were acquired using an inclusion list over m/z 150–2000, with a 20.0 m/z isolation window and a resolution of 140,000. In negative-ion mode, the AGC target and maximum injection time were 3e6 and 3000 ms, respectively; in positive-ion mode, they were 5e4 and 650 ms, respectively. PRM spectra were acquired using an inclusion list with a default charge state of 1 and one microscan per scan. For the standard PRM method in both polarities, the isolation window was 1.0 m/z, the resolution was 70,000, the AGC target was 5e4, the maximum injection time was 400 ms, and the normalized collision energy was 30 in negative-ion mode and 15 in positive-ion mode. An additional positive-ion PRM method was used with a 0.4 m/z isolation window, a fixed first mass of 300.0 m/z, a resolution of 140,000, an AGC target of 1e5, a maximum injection time of 3000 ms, two microscans, and a normalized collision energy of 12.5. In-source collision-induced dissociation was set to 0.0 eV for all experiments, and spectra were recorded in profile mode.
Lock mass calibration was performed using CUDA (C19H36N2O3) at m/z 339.26532 ([M-H]-) and m/z 341.27987 ([M+H]+). PE species were assessed in negative ion mode measurements and ceramide and PC were quantified from positive ion mode measurements.
"],"metabolite_name":["PC(16:0/18:1)","Cer(d18:1/18:0)"],"additional_accession":[]},"is_claimable":false,"name":"Membrane lipid as cofactor of cargo uptake and release by box-like lipid transfer proteins","description":"Lipid transfer proteins (LTPs) shuttle their cargo lipids between cellular membranes. LTPs facilitate the costly extraction of a lipid from a donor membrane, and its delivery into the acceptor membrane, but the mechanisms remain poorly understood. We combined extensive molecular dynamics simulations and biochemical assays to investigate the box-like ceramide transfer protein STARD11 and other members of the STARD family. We show that a family-wide conserved arginine in the gate region is the keystone of a phospholipid binding site which, when occupied by a phosphate group, stabilizes an open gate that induces local disruption of the membrane packing, as evidenced by upward displacement of lipid acyl chains towards the membrane interface. Mutation of this residue slows down in vitro ceramide uptake by 20-fold, whereas other mutations in the gate region have little effect. UV-induced cross-linking experiments with liposomes containing a bifunctional phosphatidylcholine show close proximity of the lipid acyl chains and STARD11WT, but not the arginine mutant. Together our results demonstrate that a membrane-resident phospholipid acts as a cofactor for efficient lipid transfer by STARD11, STARD4 and STARD1. Simulations of STARD2, STARD10 and STARD13 support a conserved structural role for this arginine, which is present in 13 STARD family members with different cargo specificity.","dates":{"publication":"2026-10-06","submission":"2026-10-06"},"accession":"MTBLS15933","cross_references":{"MetaboLights":["MTBLC73007","MTBLC16038","MTBLC72961","MTBLC73001"],"ChEBI":["CHEBI:73007","CHEBI:16038","CHEBI:72961","CHEBI:73001"]}}