Dual cationic and anionic metabolite analysis in a single cell
Dual cationic–anionic profiling of metabolites in a single identified cell in a live Xenopus laevis embryo by microprobe CE-ESI-MS
Erika P. Portero and Peter Nemes
Analyst. 2019 Jan 28;144(3):892-900. doi: 10.1039/c8an01999a.
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In situ capillary microsampling with capillary electrophoresis (CE) electrospray ionization (ESI) mass spec- trometry (MS) enabled the characterization of cationic metabolites in single cells in complex tissues and organisms. For deeper coverage of the metabolome and metabolic networks, analytical approaches are needed that provide complementary detection for anionic metabolites, ideally using the same instrumen- tation. Described here is one such approach that enables sequential cationic and anionic (dual) analysis of metabolites in the same identified cell in a live vertebrate embryo. A calibrated volume was microaspirated from the animal-ventral cell in a live 8-cell embryo of Xenopus laevis, and cationic and anionic metab- olites were one-pot microextracted from the aspirate, followed by CE-ESI-MS analysis of the same extract. A laboratory-built CE-ESI interface was reconfigured to enable dual cationic–anionic analysis with ∼5–10 nM (50–100 amol) lower limit of detection and a capability for quantification. To provide robust separation and efficient ion generation, the CE-ESI interface was enclosed in a nitrogen gas filled chamber, and the operational parameters were optimized for the cone-jet spraying regime in both the posi- tive and negative ion mode. A total of ∼250 cationic and ∼200 anionic molecular features were detected from the cell between m/z 50–550, including 60 and 24 identified metabolites, respectively. With only 11 metabolites identified mutually, the duplexed approach yielded complementary information on metab- olites produced in the cell, which in turn deepened network coverage for several metabolic pathways. With scalability to smaller cells and adaptability to other types of tissues and organisms, dual cationic–anionic detection with in situ microprobe CE-ESI-MS opens a door to better understand cell metabolism.
Fig. 1 Microprobe CE-ESI-MS strategy to measure cationic and anionic metabolites from the same identified cell in a live X. laevis embryo. Shown here, the left animal-ventral (V1) cell of the 8-cell embryo was identified, and ∼10 nL of its content was aspirated for one-pot metabolite extraction, followed by cationic and anionic profiling of the same cell extract. Scale bars = 250 μm.
Fig. 3 Cationic and anionic profiling of metabolites in the same V1 cell in a live X. laevis embryo. Representative extracted ion electropherograms are shown for select signals. Identified metabolites are labeled (see abbreviations in Tables S2 and 3†). Numbers correspond to molecular features in Table S4.
Fig. 5 KEGG pathway analysis for metabolites identified in single V1 cells. Values of statistical significance (p) and impact are shown for labeled pathways in Table 1. Pathway view for arginine–proline metab- olism marking complementary detection by cationic and anionic ana- lyses. Key: ASA, argininosuccinate; Cit, citrulline; CR, creatine; Gly-Phos, Glycero-phospholipid metabolism; Hyp, hydroxyproline; P-CR, phos- phocreatine; SPM, spermidine; V6B, vitamin 6B metabolism.
Adapted with permission from Royal Society of Chemistry: Portero & Nemes (2019). Dual cationic–anionic profiling of metabolites in a single identified cell in a live Xenopus laevis embryo by microprobe CE-ESI-MS. Analyst January 28, 2019; 144 (3): 892-900. Published by Royal Society of Chemistry. doi: 10.1039/c8an01999a. Copyright (2019).Last Updated: 2019-02-19