Mass spectrometric studies of the path of carbon in photosynthesis: positional isotopic analysis of ¹³C‐labelled C₄ to C₇ sugar phosphates

Abstract

The (EIMS) electron ionization mass spectrometric fragmentation patterns of the methoxime- and ethoxime-trimethylsilyl (TMS) derivatives of C(4) to C(7) sugars involved as phosphates in the Calvin pathway of photosynthesis in plants were analysed by gas chromatography/EIMS using specifically labelled (13)C analogs. In general, most but not all of the major ions in the mass spectra arise from single carbon-carbon bond cleavages of the straight-chain derivatives. The results confirm that GC/MS of the alkoxime-TMS derivatives is a viable method for measuring (13)C incorporations at individual carbon atoms in each of the sugar phosphates during photosynthetic experiments with (13)CO(2).

Metabolic Fluxes in an Illuminated Arabidopsis Rosette

“…Analytes were manually identified using the TagFinder plug-in of the TagFinder software (Luedemann et al, 2008) and the reference library mass spectra and retention indices housed in the Golm Metabolome Database (http://gmd.mpimp-golm.mpg.de; Kopka et al, 2005). Interpretation of gas chromatography-electron impact fragments derived in GC-TOF-MS analysis were performed based on previously published detailed EI fragmentation pattern of trimethylsilylated and methoxiaminated carbohydrates (DeJongh et al, 1969;Laine and Sweeley, 1973;MacLeod et al, 2001;Sanz et al, 2002) and amino acids (Abramson et al, 1974;Leimer et al, 1977). Furthermore, interpretation was supported by the mass shifts observed upon in vivo 13 C labeling (Huege et al, 2007).…”

“…In the last 10 years, new analytical chemistry tools, like metabolomics, have increased our knowledge of metabolism (Nunes-Nesi et al, 2005;Arrivault et al, 2009;Stitt et al, 2010). The use of stable isotopes (Schaefer et al, 1980;MacLeod et al, 2001;Schwender et al, 2004a;Römisch-Margl et al, 2007), coupled with improvements in analytical methods, has opened new possibilities for metabolism research (DellaPenna and Last, 2008;Saito and Matsuda, 2010).…”

GC-EI-TOF-MS analysis of in vivo carbon-partitioning into soluble metabolite pools of higher plants by monitoring isotope dilution after 13CO2 labelling

“…Other cases of potentially ''non-unique'' origin, also indicated in Supplementary file 1, can only be recognized and verified after detailed analysis of positional labelled metabolites (e.g. MacLeod et al, 2001).…”

“…1). This interpretation effort was based on the detailed EI fragmentation patterns of trimethylsilylated and methoxiaminated carbohydrates (DeJongh et al, 1969;Laine and Sweeley, 1973;MacLeod et al, 2001;Sanz et al, 2002) or amino acids (Abramson et al, 1974;Bergströ m et al, 1970;Leimer et al, 1977), which have been previously published. Furthermore, interpretation was supported by the mass shifts observed upon in vivo 13 C-labelling ( Fig.…”

“…Calvin, 1956Calvin, , 1964 or stable isotope tracing (e.g. Schaefer et al, 1975Schaefer et al, , 1980MacLeod et al, 2001;Schwender et al, 2004) has been used since decades utilizing the main entry points of CO 2 into plant metabolism, namely ribulose-1,5-diphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31). Over time, CO 2 tracing yielded ground-breaking biological insights into photosynthetic carbon assimilation, photorespiration and metabolism and, thus, into essential life-sustaining physiological mechanisms on earth.…”