Section 3 (analysis of targeted lipidomics datasets – SRM, MRM, PRM)

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capture_3 OzFAD Skyline LipidCreator METLIN-MRM/XCMS-MRM
Analytical strategies Fragmentation is by ion-ion reaction of ozone with unsaturated fatty acyls, leading to diagnostic product ions. Custom and automated definition of transitions is dependent on the input raw dataset from the DIA LC-OzID-MS acquisition. Library matching SRM, MRM, PRM, DIA SRM, MRM
Covered lipid subclasses FA; — PL, GL, SP, ST, FA 8 out of 8 LIPID MAPS categories
Number of transitions For LC-OzID-MS/MS DDA, typically 2k transitions or targets (entries in inclusion list) are automatically defines based on the processing of the LC-OzID-MS DIA dataset. Transitions in Skyline within automated processing steps can reach 500000 for comprehensive analysis of natural samples in full discovery mode. No limit On-fly/on-demand transition generation 32,347
Number of experimentally optimized transitions — — — —
Number of computationally optimized transitions — — — —
Transitions for different collision energies None, CID is not the fragmentation technique employed Yes Yes Yes
Peak detection/alignment A combination of Skyline and python processing is used to ensure peak detection and alignment. Quantification of peaks can be manually verified and adjusted in Skyline while running the workflow. Yes Via Skyline By XCMS-MRM module
Spectra annotation Algorithms for several data analysis steps are written in python and published on the github repository — LipidCreator generate in-silico spectral libraries and assays for targeted lipidomics analysis; requires Skyline (https://skyline.ms/project/home/begin.view) for the information for identification and quantification. METLIN-MRM provide optimized MRM transitions for acquisition, and XCMS-MRM use the data for targeted analysis
Quantification Validated against small subset of 37 analytical standards as well as NIST 1950 SRM original sample and literature values. Quantification for newly discovered fatty acids is a sophisticated best estimate. Yes Relative and absolute (support building calibration curves) —

* DIA – data-independent acquisition; PRM – parallel reaction monitoring; MRM – multiple reaction monitoring; SRM – single reaction monitoring