LC-MS-Based Targeted Metabolomics for FACS-Purified Rare Cells

Abstract

Metabolism plays a fundamental role in regulating cellular functions and fate decisions. Liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomic approaches provide high-resolution insights into the metabolic state of a cell. However, the typical sample size is in the order of 10 5 −10 7 cells and thus not compatible with rare cell populations, especially in the case of a prior flow cytometry-based purification step. Here, we present a comprehensively optimized protocol for targeted metabolomics on rare cell types, such as hematopoietic stem cells and mast cells. Only 5000 cells per sample are required to detect up to 80 metabolites above background. The use of regularflow liquid chromatography allows for robust data acquisition, and the omission of drying or chemical derivatization avoids potential sources of error. Cell-type-specific differences are preserved while the addition of internal standards, generation of relevant background control samples, and targeted metabolite with quantifiers and qualifiers ensure high data quality. This protocol could help numerous studies to gain thorough insights into cellular metabolic profiles and simultaneously reduce the number of laboratory animals and the time-consuming and costly experiments associated with rare cell-type purification.

Developmental regulation of primitive erythropoiesis

“…We are only beginning to understand the metabolic changes that occur as primitive erythroid cells differentiate. Current metabolomics approaches will need to be optimized for small cell numbers [82] and be supplemented by imaging strategies that will allow metabolic states to be visualized in vivo, including contributions by the niche. Taken together, the recent studies outlined here point to the complexity of regulation at transcriptional, epigenetic and metabolic levels and point to an increasing need in the future to integrate this new knowledge to better understand the emergence and differentiation of primitive erythroid cells.…”

Bidirectional interplay between metabolism and epigenetics in hematopoietic stem cells and leukemia

“…DeVilbiss et al ( 2021 ) developed a new method involving hydrophilic liquid interaction chromatography (HILIC) and high‐sensitivity orbitrap mass spectrometry (MS) that detects 160 metabolites in only 10,000 mouse HSCs. Using an alternative HILIC‐MS approach and comparable cell numbers, Schönberger et al provided insights into the metabolic differences between mouse HSCs and their immediate downstream multipotent progenitors (Schönberger et al , 2022a , b , 2023 ). With these established methodologies, it would be now interesting to address the metabolic fingerprints of human HSCs and LSCs.…”