The molecular mechanism of on-demand sterol biosynthesis at organelle contact sites-4

Created by https://lipidomicstandards.org, version v2.5.0 Contents of Report Direct Infusion Workflow 1 Overall study design ...................................................... 1 Lipid extraction ......................................................... 1 Analytical platform ....................................................... 1 Quality control ......................................................... 1 Method qualification and validation .............................................. 2 Reporting ............................................................ 2 Sample Descriptions 2 Control RNAi/BAP31 RNAi/Control plasmid/OE BAP31 / Human / Cells ......................... 2 Lipid Class Descriptions 2 1) ST[M+NH4]+ / Lipid identification ............................................ 2 1) ST[M+NH4]+ / Lipid quantification ............................................ 3 Direct Infusion Workflow Overall study design Title of the study The molecular mechanism of on-demand sterol biosynthesis at organelle contact sites-4 Document creation date 10/20/2025 Principal investigator Britta Brügger / Heidelberg Lipidomics Institution Heidelberg University Biochemistry Center (BZH) Corresponding Email britta.b[email protected] heidelberg.de Is the workflow targeted or untargeted? Untargeted Clinical No Lipid extraction Extraction method 2-phase system pH adjustment Hydrochloric acid 2-phase system Bligh&Dyer Were internal standards added prior extraction? Yes Analytical platform Ionizaton additives Ammonium formate Detector Mass spectrometer MS type Orbitrap MS vendor Thermo Direct type Chip MS Level MS1, MS2 Mass resolution for detected ion at MS1 High resolution Resolution at m/z 200 at MS1140000 Mass accuracy in ppm at MS13 Recording mode of raw data at MS1 Profile mode Mass window for precursor ion isolation (in Da total isolation window) 0.5 Mass resolution for detected ion at MS2 High resolution Resolution at m/z 200 at MS2140000 Mass accuracy in ppm at MS23 Recording mode of raw data at MS2 Profile mode Was/Were additional dimension/techniques used No Quality control Blanks Yes Type of Blanks Internal standard blank Quality control No 1 Method qualification and validation Method validation No Reporting Are reported raw data uploaded into repository? Available on request Are metadata available? Available on request Raw data upload Available on request Additional comments Raw data will be uploaded to MetaboLights Sample Descriptions Control RNAi/BAP31 RNAi/Control plasmid/OE BAP31 / Human / Cells Storage and collection conditions Available Provided preanalytical information Time to freeze, Storage time (month), Freeze-thaw cycles, Preservation method Temperature handling original sample 4-8 °C Instant sample preparation No Time to freeze between 10 and 60 min Snap freezing in liquid N2 Yes Storage temperature -80 °C Storage time (month) 0 Freeze-thaw cycles 1 Additives None Were samples stored under inert gas? No Additional preservation methods No Biobank samples No Lipid Class Descriptions 1) ST[M+NH4]+ / Lipid identification Lipid class ST MS Level for identification MS1, MS2 Identification level Molecular species level MS1adduct [M+NH4]+ Isotope correction at MS1Type 2 MS2adduct [M]+ Fragments for identification Fragment name -FA 2:0(+HO)-Cholesterol(35) Isotope correction at MS2Type 2 MS1verified by standard No MS2verified by standard No Background check at MS1Yes Background check at MS2Yes Did you presume assumptions for identification? No Limit of detection No Lipid Identification Software LipidXplorer Data manipulation Lock mass correction, Background subtraction Nomenclature for intact lipid molecule No Nomenclature for fragment ions No Further identification remarks Derivatization of sterols with acetyl chloride to sterol acetate. 2 1) ST[M+NH4]+ / Lipid quantification Quantitative No Normalization to reference No Batch correction No 3