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Capillary electrophoresis coupled to mass spectrometry (CE-MS) has emerged as a powerful analytical technique with significant implications for clinical research and diagnostics. The integration of information from CE and MS strengthens confidence in the identification of compounds present in clinical samples. The ability of CE to separate molecules based on their electrophoretic mobility coupled to MS enables the accurate identification and quantification of analytes, even in complex biological matrices such as human plasma.Here, we present a detailed protocol for an untargeted metabolomics study using CE-MS and its application in a study on human plasma from patients suffering Long COVID syndrome. The protocol ranges from sample preparation to biological interpretation, detailing a workflow enabling the analysis of cationic and anionic compounds, metabolite identification, and data processing., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Rhizobium leguminosarum bv. viciae ( Rlv ) UPM791 effectively nodulates pea and lentil, but bacteroids contain a number of proteins differentially expressed depending on the host. One of these host-dependent proteins (C189) is similar to a d i a mino b utyr a te-2-oxoglutarate a mino t ransferase (DABA-AT). DABA-AT activity was demonstrated with cell extracts and with purified protein, so C189 was renamed as Dat. The dat gene was strongly induced in the central, active area of pea nodules, but not in lentil. Mutants defective in dat were impaired in symbiotic performance with pea plants, exhibiting reduced shoot dry weight, smaller nodules, and a lower competitiveness for nodulation. In contrast, there were no significant differences between mutant and wild-type in symbiosis with lentil plants. A comparative metabolomic approach using cell-free extracts from bacteroids induced in pea and lentil showed significant differences among the strains in pea bacteroids whereas no significant differences were found in lentil. Targeted metabolomic analysis revealed that the dat mutation abolished the presence of 2,4-diaminobutyrate (DABA) in pea nodules, indicating that DABA-AT reaction is oriented toward the production of DABA from L-aspartate semialdehyde. This analysis also showed the presence of L-homoserine, a likely source of aspartate semialdehyde, in pea bacteroids but not in those induced in lentil. The dat mutant showed impaired growth when cells were grown with L-homoserine as nitrogen source. Inclusion of DABA or L-homoserine as N source suppressed pantothenate auxotropy in Rlv UPM791, suggesting DABA as source of the pantothenate precursor β-alanine. These data indicate that Rlv UPM791 Dat enzyme is part of an adaptation mechanism of this bacterium to a homoserine-rich environment such as pea nodule and rhizosphere., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ballesteros-Gutiérrez, Albareda, Barbas, López-Gonzálvez, Dunn and Palacios.)

Capillary electrophoresis coupled to mass spectrometry is an analytical platform ideal for the analysis of ionic or polar metabolites. It constitutes a perfect complement to reversed-phase liquid chromatography, offering a good alternative to polar stationary phases where reproducibility is not guaranteed. Herein, we describe a robust standardized methodology for the fingerprinting analysis of Leishmania, a taxonomic genus which comprises more than 20 protozoa species.

A lack of viable hits, increasing resistance, and limited knowledge on mode of action is hindering drug discovery for many diseases. To optimize prioritization and accelerate the discovery process, a strategy to cluster compounds based on more than chemical structure is required. We show the power of metabolomics in comparing effects on metabolism of 28 different candidate treatments for Leishmaniasis (25 from the GSK Leishmania box, two analogues of Leishmania box series, and amphotericin B as a gold standard treatment), tested in the axenic amastigote form of Leishmania donovani. Capillary electrophoresis-mass spectrometry was applied to identify the metabolic profile of Leishmania donovani, and principal components analysis was used to cluster compounds on potential mode of action, offering a medium throughput screening approach in drug selection/prioritization. The comprehensive and sensitive nature of the data has also made detailed effects of each compound obtainable, providing a resource to assist in further mechanistic studies and prioritization of these compounds for the development of new antileishmanial drugs.