Multiplexed and single cell tracing of lipid metabolism

Abstract

Cellular lipid metabolism is a complex network process comprising dozens of enzymes, multiple organelles and more than a thousand lipid species. Tracing metabolic reactions in this network is a major technological and scientific challenge. Using a click-chemistry mass spectrometry reporter strategy, we have developed a specific, highly sensitive and robust tracing procedure for alkyne-labeled lipids. The method enables sample multiplexing, which improves sample comparison. We demonstrate this by a time-resolved analysis of hepatocyte glycerolipid metabolism with parallel quantitative monitoring of 120 labeled lipid species. The subfemtomole sensitivity enabled a single cell analysis of fatty acid incorporation into neutral and membrane lipids. The results demonstrate the robustness of lipid homeostasis at the single cell level.

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Fig. 1: Structure and functioning of the click-MS reporters.
Fig. 2: Multiplexed metabolic tracing.
Fig. 3: Lipid class level analysis of the experiment outlined in Fig. 2b.
Fig. 4: Fatty acid remodeling of glycerolipids labeled with aPal.
Fig. 5: Fatty acid remodeling of glycerolipids labeled with aLino.
Fig. 6: Single cell fatty acid tracing analyzed on the lipid class level.

Data availability

With the accession https://doi.org/10.22000/251 we have published a data archive that contains all MS spectra .raw files and the corresponding LipidXplorer masterscan .sc files on which the figures are based, organized per figure or supplementary item.

Code availability

In the data archive, we have included the LipidXplorer .mfql files that were used for the extraction of identifications from the .sc files.

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Acknowledgements

We gratefully acknowledge financial support from the Deutsche Forschungsgemeinschaft (German Research Foundation) under Germany’s Excellence Strategy (grant nos. EXC2151-390873048). We thank M. Yaghmour for excellent technical assistance and L. Kuerschner for helpful comments to the manuscript.

Author information

Affiliations

Authors

Contributions

C.T. developed the concept of the project, performed the chemical synthesis and characterization, performed a part of the labeling experiments, wrote the .mfql search files, performed the data analysis, prepared the figures and wrote the manuscript. K.W. prepared the hepatocytes and performed labeling experiments and data analysis. P.L. contributed to the development of the labeling strategy, performed a part of the mass spectrometric analysis and maintained the MS instrument.

Corresponding author

Correspondence to Christoph Thiele.

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Competing interests

The corresponding author’s institution, Rheinische Friedrich-Wilhelms-Universität Bonn, has filed the patent application EP 18180252 that covers the C171 and C175 reagents as well as the concept of multiplexed lipid tracing.

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Peer review information Nicole Rusk was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 MS1 overview spectrum of hepatocytes labeled with alkyne fatty acid, before the click reaction.

Freshly isolated hepatocytes were incubated with 100 µM aPal for 2h. Cells were washed, lipids extracted and subjected to MS before click-reaction with C171 to obtain overview MS1 spectra from m/z 300-1200. Major unlabeled normal cellular lipid species are labeled in black, major peaks containing labeled species in red. The experiment was repeated 3 times with similar results.

Supplementary Figure 2 MS1 overview spectrum of hepatocytes labeled with alkyne fatty acid, after the click reaction.

Freshly isolated hepatocytes were incubated with 100 µM aPal for 2h. Cells were washed, lipids extracted and subjected to MSafter click-reaction with C171 to obtain overview MS1 spectra from m/z 300-1200. Major unlabeled normal cellular lipid species are labeled in black, major peaks containing labeled species in red. Red bars indicate the typical m/z ranges of major labeled classes, which fall into regions that are not densely populated by endogenous lipids. The experiment was repeated 3 times with similar results.

Supplementary Figure 3 Fragmentation pattern of alkyne-labeled neutral lipids click-reacted to C171.

Synthetic lipids as indicated were mixed, reacted with C171 and analyzed in MS2 at different collision energies of NCE 10 and NCE35. The experiment was repeated 12 times with similar results.

Supplementary Figure 4 Fragmentation pattern of alkyne labeled phospholipids click-reacted to C171.

Synthetic lipids as indicated were mixed, reacted with C171 and analyzed in MS2 at different collision energies of NCE 10 and NCE35. The experiment was repeated 12 times with similar results.

Supplementary Figure 5 Sensitivity and linearity of mass spectrometric analysis of alkyne lipids clicked to C171.

The internal standard mixture containing 10 different deuterium-labeled alkyne lipids in known amounts was used to prepare a dilution series that was subjected to click reaction with C171 and subsequent mass spectrometric analysis, followed by automated peak identification using LipidXplorer software. (a-e) The intensity as delivered by the software is plotted against the absolute amount of substance in the sample (final sample volume in the MS analysis: 200 µl). (f) Slope, intercept and R2 values are obtained from linear regression analysis of all individual data points. For all determinations, n=6. The experiment was repeated 3 times with similar results.

Supplementary Figure 6 Incorporation of aLino into hepatocyte lipids analyzed on the lipid class level.

Freshly isolated hepatocytes were pulse-labeled with aLino for 5 min followed by chases for 0-40 min as indicated. Lipid extracts were clicked with C175 reagents, pooled and analyzed by multiplexed mass spectrometry. Signals were identified using LipidXplorer analysis and quantified relative to signals of internal standards. Total incorporation over all analyzed classes (a) is shown in pmol, relative amounts (b) as mol% of the labeled classes. Changes in weighed average class chain lengths (c) and number of double bonds (d) indicate species remodeling within a lipid class. Note that the number of double bonds in (d) does not contain the triple bond(s) of the alkyne fatty acid used for labeling. The bars represent the mean of the three replicas, the individual values are shown as black symbols. The experiment was repeated 3 times with similar results.

Supplementary Figure 7 Mass spectrometric analysis of aPal incorporation into lipids of a single hepatocyte.

Spectra of labeled PC, TAG and double-labeled TAG of a bona-fide single hepatocyte. All numbers are fmol/sample. The experiment was repeated 3 times with similar results.

Supplementary Figure 8 Mass spectrometric analysis of aPal incorporation into lipids of multiple single hepatocytes in mole% of total.

Spectra of 6 out of 19 individual single cells collected at nominal dilutions of 0.8 cells per sample. In addition, the average of all cells in the experiment (all cells) is shown. Data indicate the fraction of the respective lipid species as mol%.relative to total labeled material. The experiment was repeated 3 times with similar results.

Supplementary Figure 9 Mass spectrometric analysis of aPal incorporation into lipids of multiple single hepatocytes as mole% of lipid classes.

Spectra of 6 out of 19 individual single cells collected at nominal dilutions of 0.8 cells per sample. In addition, the average of all cells in the experiment (all cells) is shown. Data indicate the fraction of the respective lipid species relative to the respective lipid class in mol%.Note that this is based on the same primary data than the representation in Supplementary Fig. 8. The experiment was repeated 3 times with similar results.

Supplementary Figure 10

Statistical analysis of the single cell experiment shown in Supplementary Figs. 79.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10.

Reporting Summary

Supplementary Protocol

Synthetic procedures

Supplementary Dataset 1

Incorporation of aPal, aOle or aLino into various lipid classes.

Supplementary Dataset 2

Annotated MS2 spectra of a multiplexed labeled sample.

Supplementary Table 1

Characteristic NL that occur in MS2.

Supplementary Table 2

Analysis of isotopic purity of the C175-XX reagent series.

Supplementary Table 3

Lipid species level analysis and lipid class level analysis of labeling with aPal (related to Figs 3 and 4).

Supplementary Table 4

Lipid species level analysis and lipid class level analysis of labeling with aLino (related to Fig. 5 and Supplementary Fig. 6).

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Thiele, C., Wunderling, K. & Leyendecker, P. Multiplexed and single cell tracing of lipid metabolism. Nat Methods 16, 1123–1130 (2019). https://doi.org/10.1038/s41592-019-0593-6

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