Presentation Information
[P04-526]Spatial and structural characterization of monoterpenoid indole alkaloids in Catharanthus roseus via integrative mass spectrometry
○Tetsuya Mori1, Noriko Takeda-Kamiya1, Keijiro Ohshimo2, Miyako Sakurai1, Reiki Yasukawa2, Mai Uzaki1, Kiminori Toyooka1, Masami Yokota Hirai1,3 (1. RIKEN (Japan), 2. Hokkaido University of Education (Japan), 3. Nagoya University (Japan))
Keywords:
mass spectrometry,quantum chemical calculations,ion mobility,spatial metabolomics,monoterpenoid indole alkaloids
[Purpose]
Plant alkaloids exhibit remarkable structural diversity and often show tissue-specific localization that reflects their biosynthesis and biological functions. Mass spectrometry imaging (MSI) enables spatially resolved metabolomic analysis; however, structural interpretation of alkaloid-related signals remains limited because of isomeric complexity and the limited discriminating power of m/z alone. To address this issue, we performed a comprehensive analysis of monoterpenoid indole alkaloids (MIAs) in Catharanthus roseus by combining MSI with ion mobility spectrometry and density functional theory (DFT)-based quantum chemical calculations.
[Method]
Cryosections of germinating seeds of C. roseus were prepared in a cryostat (CM3050S, Leica Microsystems) for MSI. A matrix solution of 2,5-dihydroxybenzoic acid was sprayed onto the sections using a TM Sprayer (HTX Technologies). The matrix-coated section was analyzed using matrix-assisted laser desorption/ionization-trapped ion mobility spectrometry-time of flight-mass spectrometry (MALDI-tims-TOF-MS, Bruker). Data analysis was performed using SCiLS Lab (Bruker) and MetaboScape (Bruker). Conformational searches and DFT calculations were performed for chemically annotated MIAs to determine their preferred conformations. Neutral MIAs were explored using CONFLEX with the MMFF94s force field, and protonated isomers were optimized at the M06-2X/cc-pVDZ level using Gaussian 16. Theoretical mobilities and collision cross sections were calculated using the trajectory method in IMoS under N2 buffer gas.
[Results]
Ion mobility-coupled MSI enabled visualization of MIAs in germinating C. roseus seeds and separation of several isomeric signals. Ajmalicine and tetrahydroalstonine, known stereoisomers in C. roseus, each exhibited more than three peaks in extracted ion mobilograms, suggesting the presence of additional isomeric or protomeric species. Multiple peaks were also observed for authentic standards, supporting the possible formation of protomers during ionization. To aid structural interpretation, quantum chemical calculations were performed for annotated alkaloids. As an initial case study, tetrahydroalstonine was examined in detail. The calculated CCS values of candidate protonated structures showed good agreement with experimental values, supporting the assignment of the observed mobility peaks to distinct protomeric forms. Similar analyses are ongoing for other alkaloids.
[Consideration]
Because C. roseus contains numerous structurally related MIA isomers, structural assignment based solely on MSI or m/z information is limited. Ion mobility-MSI provides additional separation, and quantum chemical calculations support the interpretation of mobility-resolved peaks.
[Conclusion]
Ion mobility–MSI visualized and separated MIA isomers, and quantum chemical calculations supported the structural interpretation of protomeric species across multiple alkaloids.
Plant alkaloids exhibit remarkable structural diversity and often show tissue-specific localization that reflects their biosynthesis and biological functions. Mass spectrometry imaging (MSI) enables spatially resolved metabolomic analysis; however, structural interpretation of alkaloid-related signals remains limited because of isomeric complexity and the limited discriminating power of m/z alone. To address this issue, we performed a comprehensive analysis of monoterpenoid indole alkaloids (MIAs) in Catharanthus roseus by combining MSI with ion mobility spectrometry and density functional theory (DFT)-based quantum chemical calculations.
[Method]
Cryosections of germinating seeds of C. roseus were prepared in a cryostat (CM3050S, Leica Microsystems) for MSI. A matrix solution of 2,5-dihydroxybenzoic acid was sprayed onto the sections using a TM Sprayer (HTX Technologies). The matrix-coated section was analyzed using matrix-assisted laser desorption/ionization-trapped ion mobility spectrometry-time of flight-mass spectrometry (MALDI-tims-TOF-MS, Bruker). Data analysis was performed using SCiLS Lab (Bruker) and MetaboScape (Bruker). Conformational searches and DFT calculations were performed for chemically annotated MIAs to determine their preferred conformations. Neutral MIAs were explored using CONFLEX with the MMFF94s force field, and protonated isomers were optimized at the M06-2X/cc-pVDZ level using Gaussian 16. Theoretical mobilities and collision cross sections were calculated using the trajectory method in IMoS under N2 buffer gas.
[Results]
Ion mobility-coupled MSI enabled visualization of MIAs in germinating C. roseus seeds and separation of several isomeric signals. Ajmalicine and tetrahydroalstonine, known stereoisomers in C. roseus, each exhibited more than three peaks in extracted ion mobilograms, suggesting the presence of additional isomeric or protomeric species. Multiple peaks were also observed for authentic standards, supporting the possible formation of protomers during ionization. To aid structural interpretation, quantum chemical calculations were performed for annotated alkaloids. As an initial case study, tetrahydroalstonine was examined in detail. The calculated CCS values of candidate protonated structures showed good agreement with experimental values, supporting the assignment of the observed mobility peaks to distinct protomeric forms. Similar analyses are ongoing for other alkaloids.
[Consideration]
Because C. roseus contains numerous structurally related MIA isomers, structural assignment based solely on MSI or m/z information is limited. Ion mobility-MSI provides additional separation, and quantum chemical calculations support the interpretation of mobility-resolved peaks.
[Conclusion]
Ion mobility–MSI visualized and separated MIA isomers, and quantum chemical calculations supported the structural interpretation of protomeric species across multiple alkaloids.
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