Fig. 2: Automated annotation of lipid standards using MS-DIAL 5 software.

a Summary of MS/MS spectra by setting 30 eV of collision energy in both positive and negative ion modes. b Calibration curve of each lipid subclass in positive and negative ion modes (n = 4). Data-dependent MS/MS mode was used for evaluation. Circles represent the linear range of the MS¹ peak with R² > 0.9800 (Supplementary Data 3). The fully filled circles represent concentrations where the C=C position could be determined, with two major fragments detected simultaneously in at least two out of four analyses. The half-filled circle for SM indicates that the C=C position was determined only on the N-acyl chain, not the long chain base. Annotation depth was assessed using the EquiSPLASH mixture containing deuterium-labeled standards. c Automated annotation results of 69 synthetic lipid standards using the optimized MS-DIAL 5 software. The UltimateSPLASH standard mixture was diluted 10, 20, 50, 100, 200, 500, 1000, and 2000 times with MeOH. For example, “x10” indicates a dilution 10 times less concentrated than the original, denoted as “x1”. Colors represent the structural depth of the automatic annotation: red indicates both fatty acyl composition and C=C position (MSL + DB); orange indicates fatty acyl composition only (MSL); green indicates species level annotation only (SL); blue indicates the misannotation of C=C position (DB_misannotation). If the MS/MS spectrum was not assigned to the precursor ion by data-dependent acquisition, a square shape with a black color is used (noMS²) The representative annotation was determined as follows: if the same lipid name was annotated in at least two of the four replicates, that name was used as the representative annotation. If the annotation results differed across all four replicates, the lipid with the highest score was adopted as the representative. In this study, fragment ions corresponding to the sphingobase Δ4 C=C positions were not detected under the applied collision energy settings. However, C=C position-specific fragment ions from N-acyl chains were detected, as confirmed using UltimateSPLASH standards. Therefore, the label “MSL + DB” was assigned to sphingolipids when the N-acyl chain C=C positions were determined. Lysophospholipids such as LPC, LPE, LPG, lyso-PI (LPI), and lyso-PS (LPS) are out of range for C=C position determination due to their saturated fatty acyl moieties. d Annotation results using the MS-DIAL 5 program when the MSL annotations were carried out before the C=C positional annotations were performed. The definitions of color and symbol are the same as used in Fig. 2c.