Single cell metabolic profiling of macrophages using 3D OrbiSIMS: correlations with phenotype
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ABSTRACT: Macrophages are important immune cells that respond to environmental cues acquiring a range of activation status represented by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at each end of their spectrum. Characterizing the metabolic signature (metabolic profiling) of different macrophage subsets is a powerful tool to understand the response of the human immune system to different stimuli. Here, the recently developed 3D OrbiSIMS instrument is applied to yield useful insight into the metabolome from individual cells after in-vitro differentiation of macrophages into naïve, M1 and M2 phenotypes using different cytokines. This analysis strategy not only requires more than 6 orders of magnitude less sample than traditional mass spectrometry approaches, but also allows the study of cell-to-cell variance. Characteristic metabolites in macrophage subsets are identified using a targeted lipid and data driven multivariate approached highlighting amino acids and other small molecules. The diamino acids alanylsparagine and lipid sphingomyelin, SM(d18/16:0) are uniquely found in M1 macrophages while pyridine and pyrimidine are observed at increased intensity in M2 macrophages, findings which link to known biological pathways. The first demonstration of this capability illustrates the great potential of direct cell analysis for in-situ metabolite profiling with the 3D OrbiSIMS to probe functional phenotype at the single cell level using molecular signatures and to understand the response of the human body to implanted devices and immune diseases.
- Secondary ion mass spectrometry
- Macrophages, 3D OrbiSIMS, PCA, Metabolomics
- Biological Sciences::Molecular biology, biophysics & biochemistry::Medical & veterinary biochemistry
- Q Science::QR Microbiology::QR180 Immunology
- University of Nottingham, UK Campus::Faculty of Science::School of Pharmacy
Data type3D OrbiSIMS
- Suvannapruk, Waraporn
- Alexander, Morgan
- Ghaemmaghami, Amir
- Scurr, David
- Edney, Max
- Engineering & Physical Sciences Research Council
Data collection methodThe sample was plunge frozen in liquid nitrogen and freeze dried over a period of 12 hours to remove water. The sample were subsequently stored in a sealed container and stored at –80 C until analysis. Prior to OrbiSIMS analysis, the sample was warmed to room temperature without opening and then loaded into the 3D OrbiSIMS instrument airlock for analysis. 3D OrbiSIMS analysis was performed using a HybridSIMS instrument (IONTOF, GmbH) with Mode 4 (single beam 20 keV Ar3000+, OrbitrapTM analyzer) of the instrument.