The molecular mechanism of on-demand sterol biosynthesis at organelle contact sites-3

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/Contro plasmid/OE BAP31 / Human / Cells ......................... 2 Lipid Class Descriptions 2 1) ST[M]+ / Lipid identification ................................................ 2 1) ST[M]+ / 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-3 Document creation date 10/20/2025 Principal investigator Britta Brügger / Heidelberg Lipidomics Institution Heidelberg University Biochemistry Center Corresponding Email britta.b[email protected] heidelberg.de Is the workflow targeted or untargeted? Targeted 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 acetate Detector Mass spectrometer MS type QTrap MS vendor SCIEX Direct type Chip MS Level MS2 Mass window for precursor ion isolation (in Da total isolation window) 1 Mass resolution for detected ion at MS2 Low resolution Resolution at MS2Unit Recording mode of raw data at MS2 Profile mode Was/Were additional dimension/techniques used No Quality control Blanks Yes Type of Blanks Extraction blank, Solvent blank, Internal standard blank Quality control Yes Type of QC sample Sample pool 1 Method qualification and validation Method validation Yes Lipid recovery Yes Dynamic quantification range No Limit of quantitation (LOQ)/Limit of detection (LOD) No Precision Yes Accuracy Yes Guidelines followed None 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/Contro 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 620 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]+ / Lipid identification Lipid class ST MS Level for identification MS2 Identification level Molecular species level MS2adduct [M]+ Fragments for identification Fragment name -FA2:0(+HO)-Cholesterol(35) Isotope correction at MS2Type 2 MS2verified by standard Yes Background check at MS2Yes Did you presume assumptions for identification? No Limit of detection No Lipid Identification Software Lipidview 1.3 Data manipulation 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]+ / Lipid quantification Quantitative Yes MS Level for quantification MS2 Internal lipid standard(s) MS2 Internal standard Fragment(s) Endogenous subclass d7-ST27:1;O -FA2:0(+HO)-d7-Cholesterol(35) Type of quantification Internal standard amount Response correction One factor for all species Type I isotope correction Yes Limit of quantification No Normalization to reference No Lipid Quantification Software LipidView 1.3 and homemade Batch correction No 3