Extended Data Fig. 7: Identification of labeled TG;Y in heavy isotope-FA labeling experiments.

3T3-L1 cells were labeled with heavy-isotope labeled FA (11:0, 16:0, 18:2, each at 50 µM) for 1 h, and lipids extracted. Spectra were recorded and, using the ion map function of the Themo Xcalibur software, fragmentation spectra were reconstructed. Panel a is the MS2 spectrum of neutral loss of 0 Da, equivalent to a total MS1 spectrum. Panel b shows the neutral loss spectrum for the fragment m/z 206.2, which is FA 11:0[D3] + NH₃, specific for TG containing FA 11:0[D3]. Comment: It is obvious that the isotope labeling data in Extended Data Fig. 7 do not reach the quality of the click-labeling in the corresponding Extended Data Fig. 6. The labeled peaks in the isotope labeling tend to disappear in the background, the intensity of the NL spectrum in panel b is only 12 % of that in the corresponding Extended Data Fig. 6c, even though this is actually a multiplex sample that contains the same information four times (additional signals at m/z NL 75.1, 76.1 and 77.1). Further, the data in Extended Data Fig. 7b are more complex than those in Extended Data Fig. 6c, because they contain not only the single-labeled species but also all the combinations of FA 11:0[D3] with itself and the other labeled fatty acids. In contrast, Extended Data Fig. 6c is a clean spectrum of TG;Y1 with FA 11:0;Y, because double- and triple-labeled species are at different ranges of the spectrum and show different fragmentation preferences.