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Metabolomic profiling of single enlarged lysosomes

Abstract

Lysosomes are critical for cellular metabolism and are heterogeneously involved in various cellular processes. The ability to measure lysosomal metabolic heterogeneity is essential for understanding their physiological roles. We therefore built a single-lysosome mass spectrometry (SLMS) platform integrating lysosomal patch-clamp recording and induced nano-electrospray ionization (nanoESI)/mass spectrometry (MS) that enables concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes. The accuracy and reliability of this technique were validated by supporting previous findings, such as the transportability of lysosomal cationic amino acids transporters such as PQLC2 and the lysosomal trapping of lysosomotropic, hydrophobic weak base drugs such as lidocaine. We derived metabolites from single lysosomes in various cell types and classified lysosomes into five major subpopulations based on their chemical and biological divergence. Senescence and carcinoma altered metabolic profiles of lysosomes in a type-specific manner. Thus, SLMS can open more avenues for investigating heterogeneous lysosomal metabolic changes during physiological and pathological processes.

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Fig. 1: Detection of single-lysosome metabolome by SLMS.
Fig. 2: Applications of SLMS in lysosomal research.
Fig. 3: Classification of lysosomal subpopulations based on single-lysosome metabolomics.
Fig. 4: Correlations between lysosomal subpopulations of MEFs, MLFs and HEK293T cells.
Fig. 5: Identification of lysosomal-subpopulation-specific metabolomic changes during cellular senescence.
Fig. 6: Identification of lysosomal-subpopulation-specific metabolomic changes in carcinoma cells.

Data availability

Mass spectrometric raw data that support the findings of this study have been deposited in the MassIVE database under the accession code MSV000087208. Metabolites were identified by matching the accurate mass with data from the HMDB (https://hmdb.ca/). Also, some metabolites were further identified by comparing MS/MS spectra obtained from population lysosomes with data from the HMDB.

Code availability

All custom code is available on GitHub at https://github.com/USTC-xlab/single-lysosome-MS.

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Acknowledgements

We thank L. Zhang (National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, USA) for valuable comments and suggestions to improve the quality of the paper. We also thank D. Ren from the University of Pennsylvania for use of the cDNA construct encoding PQLC2. We acknowledge support from National Natural Science Foundation of China (grants 91849206, 91649121, 91942315 and 92049304 to W.X.; 31722018 to C.C.; 21974130, 81701068 and 91849116 to H.Z.), the National Key R&D Program of China (2016YFC1300500-2, 2020YFA0112203), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant XDB39000000), the Key Research Program of Frontier Science (CAS, grant ZDBS-LY-SM002), the Youth Innovation Promotion Association CAS (2021453 to H.Z.), the CAS Interdisciplinary Innovation Team (JCTD-2018-20), the Fundamental Research Funds for the Central Universities, the Major Program of Development Foundation of Hefei Center for Physical Science and Technology (2017FXZY006) and the Users with Excellence Program/Project of Hefei Science Center CAS (2019HSC-UE006), USTC Research Funds of the Double First-Class Initiative (YD9100002001 to W.X. and YD9100002005 to H.Z.).

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W.X. and C.C. designed research and supervised the project; H.Z. and Q.L. performed experiments with assistance from X.Y., M.D., W.Z. and G.H.; H.Z. and T.L. analyzed data with assistance from L.Q., Q.C., Z.W., L.Y., S.G., C.M. and Z.J.; W.X., H.Z. and C.C. wrote the manuscript.

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Correspondence to Chunlei Cang or Wei Xiong.

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The authors declare no competing interests.

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Peer review information Nature Methods thanks Zhibo Yang, Mioara Larion and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Arunima Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Zhu, H., Li, Q., Liao, T. et al. Metabolomic profiling of single enlarged lysosomes. Nat Methods 18, 788–798 (2021). https://doi.org/10.1038/s41592-021-01182-8

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