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Monitoring Ci assimilation rates <jats:italic>in vivo<\/jats:italic> is a major challenge in analyzing photoautotrophic metabolism and engineering improved photosynthesis, as conventional methods do not distinguish between these two fluxes.<\/jats:p><\/jats:list-item>\n\n<jats:list-item><jats:p>We explored widely applied gas chromatography mass spectrometry (GC\u2010MS) metabolite profiling for C\u2010positional fractional <jats:sup>13<\/jats:sup>C enrichment (E<jats:sup>13<\/jats:sup>C) analyses of aspartate to differentiate within one molecule between PEPC, RUBISCO, and CBB cycle activities by <jats:sup>13<\/jats:sup>C pulse\u2010labeling. We validated this method using two GC\u2010MS instruments and two prevailing chemical derivatization methods.<\/jats:p><\/jats:list-item>\n\n<jats:list-item><jats:p>We selectively determined E<jats:sup>13<\/jats:sup>C at each carbon position of aspartate with accuracy &lt;\u20091% and precision &lt;\u20092.5%. In combination with dynamic <jats:sup>13<\/jats:sup>CO<jats:sub>2<\/jats:sub> labeling of <jats:italic>Synechocystis<\/jats:italic> cultures, we determined PEPC activity <jats:italic>in vivo<\/jats:italic> alongside assessments of RUBISCO and CBB cycle activities. We demonstrate that RUBISCO is inactive in the dark, whereas PEPC remains active but at a lower rate than during the day.<\/jats:p><\/jats:list-item>\n\n<jats:list-item><jats:p>Accurate quantifications of aspartate concentrations and positional E<jats:sup>13<\/jats:sup>Cs provide molar Ci assimilation rates of photoautotrophic <jats:italic>Synechocystis<\/jats:italic> cultures. This technology can be combined with C\u2010positional analyses of other metabolites, for example 3\u2010phosphoglycerate, and may be adapted to characterize natural and biosynthetically engineered Ci\u2010assimilation.<\/jats:p><\/jats:list-item>\n<\/jats:list>\n<\/jats:p>","DOI":"10.1111\/nph.70412","type":"journal-article","created":{"date-parts":[[2025,7,25]],"date-time":"2025-07-25T06:03:51Z","timestamp":1753423431000},"page":"401-414","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Positional <scp><sup>13<\/sup>C<\/scp> enrichment analysis of aspartate determines <scp>PEPC<\/scp> activity <i>in vivo<\/i>"],"prefix":"10.1111","volume":"248","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3249-0441","authenticated-orcid":false,"given":"Luisa","family":"Wittemeier","sequence":"first","affiliation":[{"name":"Max\u2010Planck Institute of Molecular Plant Physiology  Am M\u00fchlenberg 1 14476 Potsdam 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