387 lines
58 KiB
XML
387 lines
58 KiB
XML
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<mods:title id="1BE34DD06F32BD38C8F4FB4C6F6DA880">Assessing specialized metabolite diversity of Alnus species by a digitized LC-MS / MS data analysis workflow</mods:title>
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<mods:namePart id="C238122DC2247D662FA29E41DF718BE9">Kang, Kyo Bin</mods:namePart>
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<mods:namePart id="1A9432643D8DC6C67355B1F882366076">Woo, Sunmin</mods:namePart>
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<mods:namePart id="2CF0CCA0DF92902D0D045FA034338C4C">Ernst, Madeleine</mods:namePart>
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<mods:namePart id="E40B115408C72E1659FD48E3E00C94F3">Nothias, Louis-Félix</mods:namePart>
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<mods:date id="D1447CD77909328B4A8A63047384598A">2020</mods:date>
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2.1. MS/MS analysis and annotation of
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specialized metabolites
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–
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/
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analysis revealed that the 15
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extracts
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are different in their specialized metabolite contents, in both a qualitative and quantitative manner (
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).
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a first step of the digitized data analysis, 531 mass features were extracted from the entire dataset by MZmine2-based preprocessing, then the feature table, metadata, and extracted
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/
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spectral.mgf files were uploaded to the
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/
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molecular networking workflow (Nothias et al., 2019).
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molecular networking organized them into a network consisting of 33 molecular families (two or more connected nodes of a graph (
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<bibRefCitation id="A2564B51FFC4B91AFD1C79ABFF35E44D" author="Nguyen, D. D. & Wu, C. H. & Moree, W. J. & Lamsa, A. & Medema, M. H. & Zhao, X. & Gavilan, R. G. & Aparicio, M. & Atencio, L. & Jackson, C. & Ballesteros, J. & Sanchez, J. & Watrous, J. D. & Phelan, V. V. & Van De Wiel, C. & Kersten, R. D. & Mehnaz, S. & De Mot, R. & Shank, E. A. & Charusanti, P. & Nagarajan, H. & Duggan, B. M. & Moore, B. S. & Bandeira, N. & Palsson, B. & Pogliano, K. & Dorrestein, P. C." pageId="1" pageNumber="2" pagination="2620" refId="ref9133" refString="Nguyen, D. D., Wu, C. H., Moree, W. J., Lamsa, A., Medema, M. H., Zhao, X., Gavilan, R. G., Aparicio, M., Atencio, L., Jackson, C., Ballesteros, J., Sanchez, J., Watrous, J. D., Phelan, V. V., Van De Wiel, C., Kersten, R. D., Mehnaz, S., De Mot, R., Shank, E. A., Charusanti, P., Nagarajan, H., Duggan, B. M., Moore, B. S., Bandeira, N., Palsson, B., Pogliano, K., Gutie r rez, M., Dorrestein, P. C., 2013. MS / MS networking guided analysis of molecule and gene cluster families. Proc. Natl. Acad. Sci. U. S. A. 110, E 2611 - E 2620. https: // doi. org / 10.1073 / pnas. 1303471110." type="book chapter" year="2013">Nguyen et al., 2013</bibRefCitation>
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)) and 268 singletons (nodes not having any molecular relatives).
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<typeStatus id="197C8802FFC4B91AFF477A60FEF4E460" box="[226,275,1553,1572]" pageId="1" pageNumber="2">types</typeStatus>
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of metadata related to spectral sources, plant species and plant parts, were visualized upon the molecular network. Both species-mapping (
|
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<figureCitation id="5EFC2A25FFC4B91AFEB47A38FEB3E418" box="[273,340,1609,1628]" captionStart="Fig" captionStartId="2.[100,130,948,965]" captionTargetBox="[188,1399,154,924]" captionTargetId="figure-712@2.[187,1400,152,925]" captionTargetPageId="2" captionText="Fig. 1. LC–MS base peak ion (BPI) chromatograms of 15 Alnus extracts. Gaps between chromatogram were added to visualize their difference, so y-axis values do not equal to the absolute intensities." figureDoi="http://doi.org/10.5281/zenodo.8294566" httpUri="https://zenodo.org/record/8294566/files/figure.png" pageId="1" pageNumber="2">Fig. S1</figureCitation>
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, Supplementary Data) and plant parts-mapping (
|
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<figureCitation id="5EFC2A25FFC4B91AFF057A14FF3DE43C" box="[160,218,1637,1656]" captionStart="Fig" captionStartId="3.[100,130,1281,1298]" captionTargetBox="[226,1361,154,1258]" captionTargetId="figure-514@3.[225,1362,152,1259]" captionTargetPageId="3" captionText="Fig. 2. The MS/MS spectral network of specialized metabolites contained in the bark, twigs, leaves, and fruits of A. japonica, A. firma, A. hirsuta, and A. hirsuta var. sibirica. Spectral nodes are colored according to the mean precursor ion intensity per different plant parts: bark, twigs, leaves, and fruits. Molecular families A–I are highlighted." figureDoi="http://doi.org/10.5281/zenodo.8294568" httpUri="https://zenodo.org/record/8294568/files/figure.png" pageId="1" pageNumber="2">Fig. 2</figureCitation>
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) visualized that many metabolites in certain molecular families are constrained to specific species plant parts. For example, spectral nodes in molecular family
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<emphasis id="F4B3EAB2FFC4B91AFE107AECFE24E4F4" bold="true" box="[437,451,1693,1712]" pageId="1" pageNumber="2">
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<collectionCode id="A0D6AE65FFC4B91AFE107AECFE24E4F4" box="[437,451,1693,1712]" country="Germany" lsid="urn:lsid:biocol.org:col:15534" name="Botanischer Garten und Botanisches Museum Berlin-Dahlem, Zentraleinrichtung der Freien Universitaet" pageId="1" pageNumber="2" type="Herbarium">B</collectionCode>
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are mainly found in fruits, while other metabolites in molecular family
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<emphasis id="F4B3EAB2FFC4B91AFE737AC8FE02E488" bold="true" box="[470,485,1721,1740]" pageId="1" pageNumber="2">D</emphasis>
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are from bark and twigs and spectra in molecular family
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<emphasis id="F4B3EAB2FFC4B91AFBE37CEEFBB4E2F6" bold="true" box="[1094,1107,159,178]" pageId="1" pageNumber="2">E</emphasis>
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are predominantly observed in leaves. Metabolites in molecular family
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<emphasis id="F4B3EAB2FFC4B91AFBD07CCAFB66E28A" bold="true" box="[1141,1153,187,206]" pageId="1" pageNumber="2">F</emphasis>
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was only found in
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<taxonomicName id="01C74D23FFC4B91AFAE97CCAFA7CE28A" box="[1356,1435,187,206]" class="Magnoliopsida" family="Betulaceae" genus="Alnus" kingdom="Plantae" order="Fagales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="firma">
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<emphasis id="F4B3EAB2FFC4B91AFAE97CCAFA7CE28A" bold="true" box="[1356,1435,187,206]" italics="true" pageId="1" pageNumber="2">A. firma</emphasis>
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</taxonomicName>
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. The largest molecular family,
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<emphasis id="F4B3EAB2FFC4B91AFB857CA6FBC8E2AE" bold="true" box="[1056,1071,215,234]" pageId="1" pageNumber="2">A</emphasis>
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, could be divided into two subclusters based on distribution (
|
||
<figureCitation id="5EFC2A25FFC4B91AFC6B7C82FBE3E342" box="[974,1028,243,262]" captionStart="Fig" captionStartId="3.[100,130,1281,1298]" captionTargetBox="[226,1361,154,1258]" captionTargetId="figure-514@3.[225,1362,152,1259]" captionTargetPageId="3" captionText="Fig. 2. The MS/MS spectral network of specialized metabolites contained in the bark, twigs, leaves, and fruits of A. japonica, A. firma, A. hirsuta, and A. hirsuta var. sibirica. Spectral nodes are colored according to the mean precursor ion intensity per different plant parts: bark, twigs, leaves, and fruits. Molecular families A–I are highlighted." figureDoi="http://doi.org/10.5281/zenodo.8294568" httpUri="https://zenodo.org/record/8294568/files/figure.png" pageId="1" pageNumber="2">Fig. 2</figureCitation>
|
||
). This finding reveals the localization of closely related yet different chemical structures in different plant parts.
|
||
</paragraph>
|
||
<paragraph id="C67836A0FFC4B91AFCF67D5AFBFAE035" blockId="1.[818,1488,159,1992]" pageId="1" pageNumber="2">
|
||
The MS/MS spectral library search through GNPS resulted in 47 hits to reference MS/MS spectra, which are level 2 annotations according to the 2007 Metabolomics Standards Initiative (MSI) (
|
||
<bibRefCitation id="A2564B51FFC4B91AFA9A7D12FC85E3D6" author="Sumner, L. W. & Amberg, A. & Barrett, D. & Beale, M. H. & Beger, R. & Daykin, C. A. & Fan, T. W. M. & Fiehn, O. & Goodacre, R. & Griffin, J. L. & Hankemeier, T. & Hardy, N. & Harnly, J. & Higashi, R. & Kopka, J. & Lane, A. N. & Lindon, J. C. & Marriott, P. & Nicholls, A. W. & Reily, M. D. & Thaden, J. J. & Viant, M. R." pageId="1" pageNumber="2" pagination="211 - 221" refId="ref10877" refString="Sumner, L. W., Amberg, A., Barrett, D., Beale, M. H., Beger, R., Daykin, C. A., Fan, T. W. M., Fiehn, O., Goodacre, R., Griffin, J. L., Hankemeier, T., Hardy, N., Harnly, J., Higashi, R., Kopka, J., Lane, A. N., Lindon, J. C., Marriott, P., Nicholls, A. W., Reily, M. D., Thaden, J. J., Viant, M. R., 2007. Proposed minimum reporting standards for chemical analysis: chemical analysis working group (CAWG) metabolomics standards initiative (MSI). Metabolomics 3, 211 - 221. https: // doi. org / 10.1007 / s 11306 - 007 - 0082 - 2." type="journal article" year="2007">Sumner et al., 2007</bibRefCitation>
|
||
). Nine previously isolated and purified diarylheptanoids, platyphylloside (
|
||
<emphasis id="F4B3EAB2FFC4B91AFC077DEBFC49E3E9" bold="true" box="[930,942,410,429]" pageId="1" pageNumber="2">1</emphasis>
|
||
; numbers mean spectra indices in the MS/MS molecular network), aceroside VII (
|
||
<emphasis id="F4B3EAB2FFC4B91AFB8E7DC7FBA3E38D" bold="true" box="[1067,1092,438,457]" pageId="1" pageNumber="2">63</emphasis>
|
||
), aceroside VIII (
|
||
<emphasis id="F4B3EAB2FFC4B91AFB527DC7FAFBE38D" bold="true" box="[1271,1308,438,457]" pageId="1" pageNumber="2">195</emphasis>
|
||
), oregonin (
|
||
<emphasis id="F4B3EAB2FFC4B91AFA397DC7FA26E38D" bold="true" box="[1436,1473,438,457]" pageId="1" pageNumber="2">202</emphasis>
|
||
), rubranoside B (
|
||
<emphasis id="F4B3EAB2FFC4B91AFC767DA3FC1FE3A1" bold="true" box="[979,1016,466,485]" pageId="1" pageNumber="2">206</emphasis>
|
||
), rubranoside A (
|
||
<emphasis id="F4B3EAB2FFC4B91AFB127DA3FB3BE3A1" bold="true" box="[1207,1244,466,485]" pageId="1" pageNumber="2">214</emphasis>
|
||
), rubranoside D (
|
||
<emphasis id="F4B3EAB2FFC4B91AFA397DA3FA26E3A1" bold="true" box="[1436,1473,466,485]" pageId="1" pageNumber="2">240</emphasis>
|
||
), (5
|
||
<emphasis id="F4B3EAB2FFC4B91AFCE37D9FFCB6E045" bold="true" box="[838,849,494,513]" italics="true" pageId="1" pageNumber="2">S</emphasis>
|
||
)–
|
||
<emphasis id="F4B3EAB2FFC4B91AFCC67D9FFC95E045" bold="true" box="[867,882,494,513]" italics="true" pageId="1" pageNumber="2">O</emphasis>
|
||
-methylhirstanonol (
|
||
<emphasis id="F4B3EAB2FFC4B91AFB907D9FFBBDE045" bold="true" box="[1077,1114,494,513]" pageId="1" pageNumber="2">241</emphasis>
|
||
), and oregonoyl A (
|
||
<emphasis id="F4B3EAB2FFC4B91AFABA7D9FFAA3E045" bold="true" box="[1311,1348,494,513]" pageId="1" pageNumber="2">246</emphasis>
|
||
) were used to confirm annotations and give level 1 annotations by matching retention time and MS/MS spectra (
|
||
<bibRefCitation id="A2564B51FFC4B91AFB8F7E57FB5AE07D" author="Lee, M. & Song, J. Y. & Chin, Y. W. & Sung, S. H." box="[1066,1213,550,569]" pageId="1" pageNumber="2" pagination="2069 - 2073" refId="ref8752" refString="Lee, M., Song, J. Y., Chin, Y. W., Sung, S. H., 2013. Anti-adipogenic diarylheptanoids from Alnus hirsuta f. sibirica on 3 T 3 - L 1 cells. Bioorg. Med. Chem. Lett. 23, 2069 - 2073. https: // doi. org / 10.1016 / j. bmcl. 2013.01.127." type="journal article" year="2013">Lee et al., 2013</bibRefCitation>
|
||
, 2010;
|
||
<bibRefCitation id="A2564B51FFC4B91AFAA37E57FA26E07D" author="Sung, S. H. & Lee, M." box="[1286,1473,550,569]" pageId="1" pageNumber="2" pagination="4648 - 4651" refId="ref11060" refString="Sung, S. H., Lee, M., 2015. Anti-adipogenic activity of a new cyclic diarylheptanoid isolated from Alnus japonica on 3 T 3 - L 1 cells via modulation of PPARγ, C / EBPα and SREBP 1 c signaling. Bioorg. Med. Chem. Lett. 25, 4648 - 4651. https: // doi. org / 10. 1016 / j. bmcl. 2015.08.032." type="journal article" year="2015">Sung and Lee, 2015</bibRefCitation>
|
||
). Retention time and spectra of these compounds can be found in Result S1, Supplementary Data.
|
||
</paragraph>
|
||
<paragraph id="C67836A0FFC4B91AFCF67E0BFC79E6FF" blockId="1.[818,1488,159,1992]" pageId="1" pageNumber="2">
|
||
To maximize the annotation coverage upon the entire dataset, we applied MolNetEnhancer, a recently developed computational workflow for MS/MS-based untargeted metabolomics (Ernst et al., 2019a). Based on the GNPS library matching and
|
||
<emphasis id="F4B3EAB2FFC4B91AFB6F7EBCFAF6E0A4" bold="true" box="[1226,1297,717,736]" italics="true" pageId="1" pageNumber="2">in silico</emphasis>
|
||
annotation derived from Network Annotation Propagation (NAP) (
|
||
<bibRefCitation id="A2564B51FFC4B91AFB5F7E98FA26E0B8" author="da Silva, R. R. & Wang, M. & Nothias, L. F. & van der Hooft, J. J. J. & Caraballo-Rodriguez, A. M. & Fox, E. & Balunas, M. J. & Klassen, J. L. & Lopes, N. P. & Dorrestein, P. C." box="[1274,1473,745,764]" pageId="1" pageNumber="2" pagination="1 - 26" refId="ref7481" refString="da Silva, R. R., Wang, M., Nothias, L. F., van der Hooft, J. J. J., Caraballo-Rodriguez, A. M., Fox, E., Balunas, M. J., Klassen, J. L., Lopes, N. P., Dorrestein, P. C., 2018. Propagating annotations of molecular networks using in silico fragmentation. PLoS Comput. Biol. 14, 1 - 26. https: // doi. org / 10.1371 / journal. pcbi. 1006089." type="journal article" year="2018">da Silva et al., 2018</bibRefCitation>
|
||
), most of the molecular families could be annotated for their unique chemical classes, which are MSI level 3 annotations. Computational class annotations made by MolNetEnhancer was double-checked manually in order to prevent false annotations. Most
|
||
<emphasis id="F4B3EAB2FFC4B91AFA8E7F28FA97E128" bold="true" box="[1323,1392,857,876]" italics="true" pageId="1" pageNumber="2">in silico</emphasis>
|
||
predicted annotations were reasonable, but some molecular families showed incorrect annotations. For example, the whole molecular family
|
||
<emphasis id="F4B3EAB2FFC4B91AFA317FE1FA44E1E7" bold="true" box="[1428,1443,912,931]" pageId="1" pageNumber="2">A</emphasis>
|
||
was annotated as triterpenoids by MolNetEnhancer, but manual inspection on molecular annotations of each spectral node suggested that two subclusters within
|
||
<emphasis id="F4B3EAB2FFC4B91AFC437F95FC12E1B3" bold="true" box="[998,1013,996,1015]" pageId="1" pageNumber="2">A</emphasis>
|
||
should be annotated as diarylheptanoids and triterpenoids, respectively (
|
||
<figureCitation id="5EFC2A25FFC4B91AFB8E7871FB96E657" box="[1067,1137,1024,1043]" captionStart="Fig" captionStartId="3.[100,130,1281,1298]" captionTargetBox="[226,1361,154,1258]" captionTargetId="figure-514@3.[225,1362,152,1259]" captionTargetPageId="3" captionText="Fig. 2. The MS/MS spectral network of specialized metabolites contained in the bark, twigs, leaves, and fruits of A. japonica, A. firma, A. hirsuta, and A. hirsuta var. sibirica. Spectral nodes are colored according to the mean precursor ion intensity per different plant parts: bark, twigs, leaves, and fruits. Molecular families A–I are highlighted." figureDoi="http://doi.org/10.5281/zenodo.8294568" httpUri="https://zenodo.org/record/8294568/files/figure.png" pageId="1" pageNumber="2">Fig. S2</figureCitation>
|
||
, Supplementary Data). Within the molecular network, class annotation elucidated the localization patterns of different classes. For example, molecular families
|
||
<emphasis id="F4B3EAB2FFC4B91AFAFA7849FA89E60F" bold="true" box="[1375,1390,1080,1099]" pageId="1" pageNumber="2">A</emphasis>
|
||
,
|
||
<emphasis id="F4B3EAB2FFC4B91AFADE7849FA6DE60F" bold="true" box="[1403,1418,1080,1099]" pageId="1" pageNumber="2">D</emphasis>
|
||
, and
|
||
<emphasis id="F4B3EAB2FFC4B91AFA677849FA2DE60F" bold="true" box="[1474,1482,1080,1099]" pageId="1" pageNumber="2">I</emphasis>
|
||
, which are present in bark and twigs, were annotated as diarylheptanoids and their glycosides. Fruits of
|
||
<taxonomicName id="01C74D23FFC4B91AFB217801FB19E6C7" box="[1156,1278,1136,1155]" pageId="1" pageNumber="2">
|
||
<emphasis id="F4B3EAB2FFC4B91AFB217801FB51E6C7" bold="true" box="[1156,1206,1136,1155]" italics="true" pageId="1" pageNumber="2">Alnus</emphasis>
|
||
species
|
||
</taxonomicName>
|
||
showed high contents of ellagitannins (
|
||
<emphasis id="F4B3EAB2FFC4B91AFC7178FDFC06E6DB" bold="true" box="[980,993,1164,1183]" pageId="1" pageNumber="2">B</emphasis>
|
||
), while the leaves were abundant in flavonoid glycosides (
|
||
<emphasis id="F4B3EAB2FFC4B91AFC2178D9FC76E6FF" bold="true" box="[900,913,1192,1211]" pageId="1" pageNumber="2">E</emphasis>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="C67836A0FFC4B919FCF678B2FD34E72A" blockId="1.[818,1488,159,1992]" lastBlockId="2.[100,770,1036,1390]" lastPageId="2" lastPageNumber="3" pageId="1" pageNumber="2">
|
||
As described in previous studies (Ernst et al., 2019a; Kang et al., 2019), the MolNetEnhacer workflow takes advantage of MS2LDA, which provides information about substructural diversity within same classes of metabolites (
|
||
<bibRefCitation id="A2564B51FFC4B91AFBB67966FAF3E76E" author="van der Hooft, J. J. J. & Wandy, J. & Barrett, M. P. & Burgess, K. E. V. & Rogers, S." box="[1043,1300,1303,1322]" pageId="1" pageNumber="2" pagination="13738 - 13743" refId="ref11138" refString="van der Hooft, J. J. J., Wandy, J., Barrett, M. P., Burgess, K. E. V., Rogers, S., 2016. Topic modeling for untargeted substructure exploration in metabolomics. Proc. Natl. Acad. Sci. U. S. A. 113, 13738 - 13743. https: // doi. org / 10.1073 / pnas. 1608041113." type="journal article" year="2016">van der Hooft et al., 2016</bibRefCitation>
|
||
). MS2LDA extracts patterns of fragment ions or neutral losses which are observed together in multiple spectra, called Mass2Motifs, which can show how the compounds in same chemical classes are different in their substructure. For example, Mass2Motifs 41, 49, 72, and 81 were extracted from MS/ MS spectra which are clustered as molecular families
|
||
<emphasis id="F4B3EAB2FFC4B91AFA8079D2FAD3E7F2" bold="true" box="[1317,1332,1443,1462]" pageId="1" pageNumber="2">A</emphasis>
|
||
,
|
||
<emphasis id="F4B3EAB2FFC4B91AFA9A79D2FAA9E7F2" bold="true" box="[1343,1358,1443,1462]" pageId="1" pageNumber="2">D</emphasis>
|
||
, and
|
||
<emphasis id="F4B3EAB2FFC4B91AFA2679D2FA6CE7F2" bold="true" box="[1411,1419,1443,1462]" pageId="1" pageNumber="2">I</emphasis>
|
||
, which are annotated as diarylheptanoids. These Mass2Motifs were annotated to represent diarylheptanoid scaffolds which are different in the pattern of unsaturation and hydroxylation. As shown in
|
||
<figureCitation id="5EFC2A25FFC4B91AFAA57987FAD1E44D" box="[1280,1334,1526,1545]" captionStart="Fig" captionStartId="4.[100,130,1441,1458]" captionTargetBox="[226,1361,153,1417]" captionTargetId="figure-274@4.[225,1362,152,1418]" captionTargetPageId="4" captionText="Fig. 3. MS2LDA-driven substructural annotation of diarylheptanoids of Alnus species. Integrated with GNPS library matching and NAP in silico annotation, diarylheptanoid-related Mass2Motifs 41, 49, 72, and 81 could be characterized and correlated with specific substructures of diarylheptanoid aglycones. Scaffold diversity within diarylheptanoid molecular families A, D, and I were revealed by mapping these Mass2Motifs on the molecular network with different colors. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8294570" httpUri="https://zenodo.org/record/8294570/files/figure.png" pageId="1" pageNumber="2">Fig. 3</figureCitation>
|
||
, Mass2Motif 72 represents the presence of a fragment ion
|
||
<emphasis id="F4B3EAB2FFC4B91AFB1F7A63FB38E461" bold="true" box="[1210,1247,1554,1573]" italics="true" pageId="1" pageNumber="2">m/z</emphasis>
|
||
331.1525 ([C
|
||
<subScript id="5A4334E5FFC4B91AFAC77A6BFA93E463" attach="both" box="[1378,1396,1562,1575]" fontSize="5" pageId="1" pageNumber="2">19</subScript>
|
||
H
|
||
<subScript id="5A4334E5FFC4B91AFA217A6BFA71E463" attach="both" box="[1412,1430,1562,1575]" fontSize="5" pageId="1" pageNumber="2">23</subScript>
|
||
O
|
||
<subScript id="5A4334E5FFC4B91AFA007A6BFA49E463" attach="left" box="[1445,1454,1562,1575]" fontSize="5" pageId="1" pageNumber="2">5</subScript>
|
||
]
|
||
<superScript id="31B29BE8FFC4B91AFA127A7FFA20E45E" attach="none" box="[1463,1479,1550,1562]" fontSize="5" pageId="1" pageNumber="2">−</superScript>
|
||
) while Mass2Motif 81 represents the presence of a fragment ion
|
||
<emphasis id="F4B3EAB2FFC4B91AFA0E7A5FFA28E405" bold="true" box="[1451,1487,1582,1601]" italics="true" pageId="1" pageNumber="2">m/z</emphasis>
|
||
299.1625 ([C
|
||
<subScript id="5A4334E5FFC4B91AFC177A23FC23E41B" attach="both" box="[946,964,1618,1631]" fontSize="5" pageId="1" pageNumber="2">19</subScript>
|
||
H
|
||
<subScript id="5A4334E5FFC4B91AFC717A23FC01E41B" attach="both" box="[980,998,1618,1631]" fontSize="5" pageId="1" pageNumber="2">23</subScript>
|
||
O
|
||
<subScript id="5A4334E5FFC4B91AFC507A23FC19E41B" attach="left" box="[1013,1022,1618,1631]" fontSize="5" pageId="1" pageNumber="2">3</subScript>
|
||
]
|
||
<superScript id="31B29BE8FFC4B91AFBA27A37FBF0E416" attach="none" box="[1031,1047,1606,1618]" fontSize="5" pageId="1" pageNumber="2">−</superScript>
|
||
), and these two Mass2Motifs are observed in MS/MS spectra
|
||
<emphasis id="F4B3EAB2FFC4B91AFC6C7A17FC09E43D" bold="true" box="[969,1006,1638,1657]" pageId="1" pageNumber="2">214</emphasis>
|
||
and
|
||
<emphasis id="F4B3EAB2FFC4B91AFBBA7A17FBDFE43D" bold="true" box="[1055,1080,1638,1657]" pageId="1" pageNumber="2">63</emphasis>
|
||
, respectively. Mass2Motif 3 represents the neural loss of
|
||
<emphasis id="F4B3EAB2FFC4B91AFC197AF3FC07E4D1" bold="true" box="[956,992,1666,1685]" italics="true" pageId="1" pageNumber="2">m/z</emphasis>
|
||
162.0525, which is caused by the loss of a hexose moiety, often related to glucose, and commonly observed in plant metabolomics data. GNPS spectral library matching annotated these spectra as rubranoside A (
|
||
<emphasis id="F4B3EAB2FFC4B91AFB8E7AA4FBB7E4AC" bold="true" box="[1067,1104,1749,1768]" pageId="1" pageNumber="2">214</emphasis>
|
||
) and aceroside VII (
|
||
<emphasis id="F4B3EAB2FFC4B91AFAB17AA4FACAE4AC" bold="true" box="[1300,1325,1749,1768]" pageId="1" pageNumber="2">63</emphasis>
|
||
). Based on these annotations, Mass2Motifs 72 and 81 could be annotated as rubranol- and centrolobol-related motifs, respectively. Similarly, Mass2Motifs 41 and 49 were annotated as hirsutanonol- and platyphyllonol-related motifs, mainly based on spectral annotation of oregonin (
|
||
<emphasis id="F4B3EAB2FFC4B91AFAD17B34FA7EE51C" bold="true" box="[1396,1433,1861,1880]" pageId="1" pageNumber="2">202</emphasis>
|
||
) and platyphylloside (
|
||
<emphasis id="F4B3EAB2FFC4B91AFC717B10FC07E530" bold="true" box="[980,992,1889,1908]" pageId="1" pageNumber="2">1</emphasis>
|
||
) (
|
||
<figureCitation id="5EFC2A25FFC4B91AFC5F7B10FBD5E530" box="[1018,1074,1889,1908]" captionStart="Fig" captionStartId="4.[100,130,1441,1458]" captionTargetBox="[226,1361,153,1417]" captionTargetId="figure-274@4.[225,1362,152,1418]" captionTargetPageId="4" captionText="Fig. 3. MS2LDA-driven substructural annotation of diarylheptanoids of Alnus species. Integrated with GNPS library matching and NAP in silico annotation, diarylheptanoid-related Mass2Motifs 41, 49, 72, and 81 could be characterized and correlated with specific substructures of diarylheptanoid aglycones. Scaffold diversity within diarylheptanoid molecular families A, D, and I were revealed by mapping these Mass2Motifs on the molecular network with different colors. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8294570" httpUri="https://zenodo.org/record/8294570/files/figure.png" pageId="1" pageNumber="2">Fig. 3</figureCitation>
|
||
). These Mass2Motifs also represent a few fragment ions with smaller
|
||
<emphasis id="F4B3EAB2FFC4B91AFBE57B0CFB82E5D4" bold="true" box="[1088,1125,1917,1936]" italics="true" pageId="1" pageNumber="2">m/z</emphasis>
|
||
values; and we confirmed that these fragment ions are identical to the previously reported characteristic fragment ions generated from each diarylheptanoid aglycone. MS/MS fragmentation pathways of diarylheptanoids under ESI negative ion mode were well established by previous studies (
|
||
<bibRefCitation id="A2564B51FFC7B919FDEA7859FF73E613" author="Riethmuller, E. & Toth, G. & Alberti, A. & Vegh, K. & Burlini, I. & Konczol, A. & Balogh, G. T. & Kery, A." pageId="2" pageNumber="3" pagination="159 - 167" refId="ref10259" refString="Riethmuller, E., Toth, G., Alberti, A., Vegh, K., Burlini, I., Konczol, A., Balogh, G. T., Kery, A., 2015. First characterisation of flavonoid- and diarylheptanoid-type antioxidant phenolics in Corylus maxima by HPLC-DAD-ESI-MS. J. Pharm. Biomed. Anal. 107, 159 - 167. https: // doi. org / 10.1016 / j. jpba. 2014.12.016." type="journal article" year="2015">Riethmüller et al., 2015</bibRefCitation>
|
||
,
|
||
<bibRefCitation id="A2564B51FFC7B919FF057835FF37E613" author="Riethmuller, E. & Alberti, A. & Toth, G. & Beni, S. & Ortolano, F. & Kery, A." box="[160,208,1092,1111]" pageId="2" pageNumber="3" pagination="493 - 503" refId="ref10183" refString="Riethmuller, E., Alberti, A., Toth, G., Beni, S., Ortolano, F., Kery, A., 2013. Characterisation of diarylheptanoid- and flavonoid-type phenolics in Corylus avellana L. leaves and bark by HPLC / DAD-ESI / MS. Phytochem. Anal. 24, 493 - 503. https: // doi. org / 10.1002 / pca. 2452." type="journal article" year="2013">2013</bibRefCitation>
|
||
), and fragment ions represented by Mass2Motifs 41 and 49 agree with the fragmentation patterns described in these studies. Thus, we could demonstrate the potential of MS2LDA, which facilitates the structural annotation of substructures and the storing of these annotations within MS2LDA experiments. These annotated Mass2Motifs can now also be stored in MotifDB (
|
||
<bibRefCitation id="A2564B51FFC7B919FE2A78A1FDD8E6A7" author="Rogers, S. & Ong, C. W. & Wandy, J. & Ridder, L. & van der Hooft, J. J. J." box="[399,575,1232,1251]" pageId="2" pageNumber="3" pagination="284 - 302" refId="ref10416" refString="Rogers, S., Ong, C. W., Wandy, J., Ernst, M., Ridder, L., van der Hooft, J. J. J., 2019. Deciphering complex metabolite mixtures by unsupervised and supervised substructure discovery and semi-automated annotation from MS / MS spectra. Faraday Discuss. 218, 284 - 302. https: // doi. org / 10.1039 / C 8 FD 00235 E." type="journal article" year="2019">Rogers et al., 2019</bibRefCitation>
|
||
). In this way, expert knowledge is made available for future substructure annotations based on MS/MS data. By mapping the distribution of Mass2Motifs on to the molecular network, we could visualize the substructural differences within molecular families (
|
||
<figureCitation id="5EFC2A25FFC7B919FECD794EFE78E716" box="[360,415,1343,1362]" captionStart="Fig" captionStartId="4.[100,130,1441,1458]" captionTargetBox="[226,1361,153,1417]" captionTargetId="figure-274@4.[225,1362,152,1418]" captionTargetPageId="4" captionText="Fig. 3. MS2LDA-driven substructural annotation of diarylheptanoids of Alnus species. Integrated with GNPS library matching and NAP in silico annotation, diarylheptanoid-related Mass2Motifs 41, 49, 72, and 81 could be characterized and correlated with specific substructures of diarylheptanoid aglycones. Scaffold diversity within diarylheptanoid molecular families A, D, and I were revealed by mapping these Mass2Motifs on the molecular network with different colors. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8294570" httpUri="https://zenodo.org/record/8294570/files/figure.png" pageId="2" pageNumber="3">Fig. 3</figureCitation>
|
||
), which is helpful during annotation and identification of MS/MS spectra within a molecular network.
|
||
</paragraph>
|
||
</subSubSection>
|
||
<footnote id="A5DC2AAEFFC4B91AFFD07A8EFF41E583" pageId="1" pageNumber="2">
|
||
<paragraph id="C67836A0FFC4B91AFFD07A8EFF41E583" blockId="1.[100,770,1791,1991]" pageId="1" pageNumber="2">
|
||
<superScript id="31B29BE8FFC4B91AFFD07A8EFF9AE54E" attach="right" box="[117,125,1791,1802]" fontSize="5" pageId="1" pageNumber="2">2</superScript>
|
||
<taxonomicName id="01C74D23FFC4B91AFF247B72FED3E550" box="[129,308,1795,1812]" class="Magnoliopsida" family="Betulaceae" genus="Alnus" kingdom="Plantae" order="Fagales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="variety" species="hirsuta" variety="sibirica">
|
||
<emphasis id="F4B3EAB2FFC4B91AFF247B72FF37E550" bold="true" box="[129,208,1795,1812]" italics="true" pageId="1" pageNumber="2">A. hirsuta</emphasis>
|
||
var.
|
||
<emphasis id="F4B3EAB2FFC4B91AFF5E7B72FED3E550" bold="true" box="[251,308,1795,1812]" italics="true" pageId="1" pageNumber="2">sibirica</emphasis>
|
||
</taxonomicName>
|
||
is currently classified as a synonym of
|
||
<taxonomicName id="01C74D23FFC4B91AFDDB7B72FD2BE550" box="[638,716,1795,1812]" class="Magnoliopsida" family="Betulaceae" genus="Alnus" kingdom="Plantae" order="Fagales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="hirsuta">
|
||
<emphasis id="F4B3EAB2FFC4B91AFDDB7B72FD2BE550" bold="true" box="[638,716,1795,1812]" italics="true" pageId="1" pageNumber="2">A. hirsuta</emphasis>
|
||
</taxonomicName>
|
||
in the Plant List (http://www.theplantlist.org). However, many local botanists are considering
|
||
<taxonomicName id="01C74D23FFC4B91AFF6A7B46FE6DE50C" box="[207,394,1847,1864]" class="Magnoliopsida" family="Betulaceae" genus="Alnus" kingdom="Plantae" order="Fagales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="variety" species="hirsuta" variety="sibirica">
|
||
<emphasis id="F4B3EAB2FFC4B91AFF6A7B46FEC7E50C" bold="true" box="[207,288,1847,1864]" italics="true" pageId="1" pageNumber="2">A. hirsuta</emphasis>
|
||
var.
|
||
<emphasis id="F4B3EAB2FFC4B91AFEF47B46FE6DE50C" bold="true" box="[337,394,1847,1864]" italics="true" pageId="1" pageNumber="2">sibirica</emphasis>
|
||
</taxonomicName>
|
||
as a
|
||
<taxonomicName id="01C74D23FFC4B91AFE1A7B46FDD5E50C" box="[447,562,1847,1864]" pageId="1" pageNumber="2" rank="variety" variety="based">variety based</taxonomicName>
|
||
on their different morphology (
|
||
<bibRefCitation id="A2564B51FFC4B91AFF107B21FEAAE525" author="Chang, K. S. & Chang, C. S. & Park, J. S." box="[181,333,1872,1889]" pageId="1" pageNumber="2" pagination="82 - 88" refId="ref7368" refString="Chang, K. S., Chang, C. S., Park, J. S., 2005. Taxonomic reconsideration of Alnus hirsuta var. hirsuta and A. hirsuta var. sibirica in Korea. Bull. Seoul Natl. Univ. Arbor. 25, 82 - 88." type="journal article" year="2005">Chang et al., 2005</bibRefCitation>
|
||
), and our study suggests that these two plants differ in their metabolite composition. Unfortunately there is no reported genome sequence of
|
||
<taxonomicName id="01C74D23FFC4B91AFF697BF2FE99E5D0" box="[204,382,1923,1940]" class="Magnoliopsida" family="Betulaceae" genus="Alnus" kingdom="Plantae" order="Fagales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="variety" species="hirsuta" variety="sibirica">
|
||
<emphasis id="F4B3EAB2FFC4B91AFF697BF2FEFDE5D0" bold="true" box="[204,282,1923,1940]" italics="true" pageId="1" pageNumber="2">A. hirsuta</emphasis>
|
||
var.
|
||
<emphasis id="F4B3EAB2FFC4B91AFEE07BF2FE99E5D0" bold="true" box="[325,382,1923,1940]" italics="true" pageId="1" pageNumber="2">sibirica</emphasis>
|
||
</taxonomicName>
|
||
which is required to confirm its taxonomy; so we concluded that this is unconcluded and kept it to be described as a genetic variety.
|
||
</paragraph>
|
||
</footnote>
|
||
<caption id="92B86628FFC7B919FFC17FC5FE9CE19A" ID-DOI="http://doi.org/10.5281/zenodo.8294566" ID-Zenodo-Dep="8294566" httpUri="https://zenodo.org/record/8294566/files/figure.png" pageId="2" pageNumber="3" startId="2.[100,130,948,965]" targetBox="[188,1399,154,924]" targetPageId="2" targetType="figure">
|
||
<paragraph id="C67836A0FFC7B919FFC17FC5FE9CE19A" blockId="2.[100,1487,948,990]" pageId="2" pageNumber="3">
|
||
<emphasis id="F4B3EAB2FFC7B919FFC17FC5FF7BE181" bold="true" box="[100,156,948,965]" pageId="2" pageNumber="3">Fig. 1.</emphasis>
|
||
LC–MS base peak ion (BPI) chromatograms of 15
|
||
<taxonomicName id="01C74D23FFC7B919FDE77FC5FD5EE181" box="[578,697,948,965]" class="Magnoliopsida" family="Betulaceae" genus="Alnus" kingdom="Plantae" order="Fagales" pageId="2" pageNumber="6" phylum="Tracheophyta" rank="species" species="extracts">
|
||
<emphasis id="F4B3EAB2FFC7B919FDE77FC5FD88E181" bold="true" box="[578,623,948,965]" italics="true" pageId="2" pageNumber="3">Alnus</emphasis>
|
||
extracts
|
||
</taxonomicName>
|
||
. Gaps between chromatogram were added to visualize their difference, so y-axis values do not equal to the absolute intensities.
|
||
</paragraph>
|
||
</caption>
|
||
<subSubSection id="8EDD652BFFC7B919FFC179D2FDF0E7F2" box="[100,535,1443,1462]" pageId="2" pageNumber="3" type="multiple">
|
||
<paragraph id="C67836A0FFC7B919FFC179D2FDF0E7F2" blockId="2.[100,535,1443,1462]" box="[100,535,1443,1462]" pageId="2" pageNumber="3">
|
||
<heading id="9D3081CCFFC7B919FFC179D2FDF0E7F2" bold="true" box="[100,535,1443,1462]" fontSize="36" level="1" pageId="2" pageNumber="3" reason="1">
|
||
<emphasis id="F4B3EAB2FFC7B919FFC179D2FDF0E7F2" bold="true" box="[100,535,1443,1462]" italics="true" pageId="2" pageNumber="3">
|
||
2.2. Chemotype discrimination of
|
||
<taxonomicName id="01C74D23FFC7B919FE3C79D2FE2CE7F2" box="[409,459,1443,1462]" class="Magnoliopsida" family="Betulaceae" genus="Alnus" kingdom="Plantae" order="Fagales" pageId="2" pageNumber="3" phylum="Tracheophyta" rank="species" species="extracts">Alnus</taxonomicName>
|
||
extracts
|
||
</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
</subSubSection>
|
||
<subSubSection id="8EDD652BFFC7B919FF2079ABFC34E555" pageId="2" pageNumber="3" type="discussion">
|
||
<paragraph id="C67836A0FFC7B919FF2079ABFDE4E5E8" blockId="2.[100,770,1498,1992]" pageId="2" pageNumber="3">
|
||
Based on the molecular network and structural annotation, chemical diversity between
|
||
<taxonomicName id="01C74D23FFC7B919FEEC7987FE2EE44D" box="[329,457,1526,1545]" class="Magnoliopsida" family="Betulaceae" genus="Alnus" kingdom="Plantae" order="Fagales" pageId="2" pageNumber="6" phylum="Tracheophyta" rank="species" species="extracts">
|
||
<emphasis id="F4B3EAB2FFC7B919FEEC7987FE9CE44D" bold="true" box="[329,379,1526,1545]" italics="true" pageId="2" pageNumber="3">Alnus</emphasis>
|
||
extracts
|
||
</taxonomicName>
|
||
were systematically analyzed. As a first step, the dissimilarity between samples was calculated. In most metabolomics studies, the diversity between samples has been analyzed by using multivariate analysis techniques such as principal component analysis (PCA) (
|
||
<bibRefCitation id="A2564B51FFC7B919FF5B7A17FE13E43D" author="Worley, B. & Powers, R." box="[254,500,1638,1657]" pageId="2" pageNumber="3" pagination="92 - 107" refId="ref12475" refString="Worley, B., Powers, R., 2012. Multivariate analysis in metabolomics. Curr. Metabolomics 1, 92 - 107. https: // doi. org / 10.2174 / 2213235 x 130108." type="journal article" year="2012">Worley and Powers, 2012</bibRefCitation>
|
||
), which is based on the Euclidean distance metric or principal coordinate analysis (PCoA) which can be based on different distance metrics such as for example the Bray-Curtis dissimilarity (
|
||
<bibRefCitation id="A2564B51FFC7B919FE957AC8FDB5E488" author="Bruckner, A. & Heethoff, M." box="[304,594,1721,1740]" pageId="2" pageNumber="3" pagination="33 - 46" refId="ref7322" refString="Bruckner, A., Heethoff, M., 2017. A chemo-ecologists' practical guide to compositional data analysis. Chemoecology 27, 33 - 46. https: // doi. org / 10.1007 / s 00049 - 016 - 0227 - 8." type="journal article" year="2017">Brückner and Heethoff, 2017</bibRefCitation>
|
||
). However, these conventional methods consider each feature as independent entities, ignoring the structural relationship between molecules. To reflect the chemical similarity at the scaffold level shared by samples from the same plant parts, we applied the Chemical Structural and Compositional Similarity (CSCS) metric (
|
||
<bibRefCitation id="A2564B51FFC7B919FE0D7B34FD9EE51C" author="Brejnrod, A. D. & Dworzynski, P. & Rasmussen, L. B. & Dorrestein, P. & van der Hooft, J. & Arumugam, M." box="[424,633,1861,1880]" pageId="2" pageNumber="3" refId="ref7251" refString="Brejnrod, A. D., Ernst, M., Dworzynski, P., Rasmussen, L. B., Dorrestein, P., van der Hooft, J., Arumugam, M., 2019. Implementations of the chemical structural and compositional similarity metric in R and Python. BioRxiv 546150. https: // doi. org / 10.1101 / 546150." type="journal volume" year="2019">Brejnrod et al., 2019</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="A2564B51FFC7B919FD2F7B34FF73E530" author="Sedio, B. E. & Echeverri, J. C. R. & Boya, C. A. & Wright, S. J." pageId="2" pageNumber="3" pagination="616 - 623" refId="ref10725" refString="Sedio, B. E., Echeverri, J. C. R., Boya, C. A., Wright, S. J., 2017. Sources of variation in foliar secondary chemistry in a tropical forest tree community. Ecology 98, 616 - 623. https: // doi. org / 10.1002 / ecy. 1689." type="journal article" year="2017">Sedio et al., 2017</bibRefCitation>
|
||
), which accounts for the chemical structural similarity across metabolites by integrating the MS/MS spectral similarity calculated in the process of GNPS molecular networking.
|
||
</paragraph>
|
||
<paragraph id="C67836A0FFC7B919FF207BC4FC34E555" blockId="2.[100,770,1498,1992]" lastBlockId="2.[818,1488,1036,1809]" pageId="2" pageNumber="3">
|
||
Compared to the PCA score plot (
|
||
<figureCitation id="5EFC2A25FFC7B919FE707BC4FDFBE58C" box="[469,540,1973,1992]" captionStart="Fig" captionStartId="5.[1129,1159,152,169]" captionTargetBox="[101,1098,152,1892]" captionTargetId="figure-1@5.[100,1099,151,1893]" captionTargetPageId="5" captionText="Fig. 4. Discrimination of the analyzed Alnus extracts into chemogroups. The analyzed extracts can be discriminated into three chemogroups by visualizing the CSCS distance metric between samples as PCoA plot (A) and chemical dendrogram (B). On the other hand, conventional methods such as PCA score plot (C) or hierarchical clustering analysis (HCA) using the Euclidean distance (D; chemogroups 1–3 are visualized with same colors used in B to make it easy to be compared) could not discriminate the samples into the same chemotypes. By mapping the chemogrouping of samples on the molecular network, it could be visualized that the three chemogroups were rich in diarylheptanoid, flavonoid, and tannins, respectively (E). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8294572" httpUri="https://zenodo.org/record/8294572/files/figure.png" pageId="2" pageNumber="3">Fig. 4C</figureCitation>
|
||
) or hierarchical cluster analysis using the Euclidean distance (
|
||
<figureCitation id="5EFC2A25FFC7B919FB0F787DFB13E65B" box="[1194,1268,1036,1055]" captionStart="Fig" captionStartId="5.[1129,1159,152,169]" captionTargetBox="[101,1098,152,1892]" captionTargetId="figure-1@5.[100,1099,151,1893]" captionTargetPageId="5" captionText="Fig. 4. Discrimination of the analyzed Alnus extracts into chemogroups. The analyzed extracts can be discriminated into three chemogroups by visualizing the CSCS distance metric between samples as PCoA plot (A) and chemical dendrogram (B). On the other hand, conventional methods such as PCA score plot (C) or hierarchical clustering analysis (HCA) using the Euclidean distance (D; chemogroups 1–3 are visualized with same colors used in B to make it easy to be compared) could not discriminate the samples into the same chemotypes. By mapping the chemogrouping of samples on the molecular network, it could be visualized that the three chemogroups were rich in diarylheptanoid, flavonoid, and tannins, respectively (E). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8294572" httpUri="https://zenodo.org/record/8294572/files/figure.png" pageId="2" pageNumber="3">Fig. 4D</figureCitation>
|
||
), the weighted (by intensity of MS1 ion intensities) CSCS metric showed clearer discriminative patterns both in a Principle Coordinates Analysis (PCoA) (
|
||
<figureCitation id="5EFC2A25FFC7B919FC9F7811FC67E637" box="[826,896,1120,1139]" captionStart="Fig" captionStartId="5.[1129,1159,152,169]" captionTargetBox="[101,1098,152,1892]" captionTargetId="figure-1@5.[100,1099,151,1893]" captionTargetPageId="5" captionText="Fig. 4. Discrimination of the analyzed Alnus extracts into chemogroups. The analyzed extracts can be discriminated into three chemogroups by visualizing the CSCS distance metric between samples as PCoA plot (A) and chemical dendrogram (B). On the other hand, conventional methods such as PCA score plot (C) or hierarchical clustering analysis (HCA) using the Euclidean distance (D; chemogroups 1–3 are visualized with same colors used in B to make it easy to be compared) could not discriminate the samples into the same chemotypes. By mapping the chemogrouping of samples on the molecular network, it could be visualized that the three chemogroups were rich in diarylheptanoid, flavonoid, and tannins, respectively (E). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8294572" httpUri="https://zenodo.org/record/8294572/files/figure.png" pageId="2" pageNumber="3">Fig. 4A</figureCitation>
|
||
) as well as the chemical dendrogram (
|
||
<figureCitation id="5EFC2A25FFC7B919FB507811FADCE637" box="[1269,1339,1120,1139]" captionStart="Fig" captionStartId="5.[1129,1159,152,169]" captionTargetBox="[101,1098,152,1892]" captionTargetId="figure-1@5.[100,1099,151,1893]" captionTargetPageId="5" captionText="Fig. 4. Discrimination of the analyzed Alnus extracts into chemogroups. The analyzed extracts can be discriminated into three chemogroups by visualizing the CSCS distance metric between samples as PCoA plot (A) and chemical dendrogram (B). On the other hand, conventional methods such as PCA score plot (C) or hierarchical clustering analysis (HCA) using the Euclidean distance (D; chemogroups 1–3 are visualized with same colors used in B to make it easy to be compared) could not discriminate the samples into the same chemotypes. By mapping the chemogrouping of samples on the molecular network, it could be visualized that the three chemogroups were rich in diarylheptanoid, flavonoid, and tannins, respectively (E). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8294572" httpUri="https://zenodo.org/record/8294572/files/figure.png" pageId="2" pageNumber="3">Fig. 4B</figureCitation>
|
||
); especially the chemodendrogram revealed that the samples can be discriminated as three chemotypes. By mapping these chemotype-classes on the molecular network, the major differences in their metabolites were easily visualized (
|
||
<figureCitation id="5EFC2A25FFC7B919FC3B78A1FC03E6A7" box="[926,996,1232,1251]" captionStart="Fig" captionStartId="5.[1129,1159,152,169]" captionTargetBox="[101,1098,152,1892]" captionTargetId="figure-1@5.[100,1099,151,1893]" captionTargetPageId="5" captionText="Fig. 4. Discrimination of the analyzed Alnus extracts into chemogroups. The analyzed extracts can be discriminated into three chemogroups by visualizing the CSCS distance metric between samples as PCoA plot (A) and chemical dendrogram (B). On the other hand, conventional methods such as PCA score plot (C) or hierarchical clustering analysis (HCA) using the Euclidean distance (D; chemogroups 1–3 are visualized with same colors used in B to make it easy to be compared) could not discriminate the samples into the same chemotypes. By mapping the chemogrouping of samples on the molecular network, it could be visualized that the three chemogroups were rich in diarylheptanoid, flavonoid, and tannins, respectively (E). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8294572" httpUri="https://zenodo.org/record/8294572/files/figure.png" pageId="2" pageNumber="3">Fig. 4E</figureCitation>
|
||
). Chemogroup 3 (Ah-L, Ahv-L, Af-L, Af-T, and Af-B) showed high contents of the molecular family
|
||
<emphasis id="F4B3EAB2FFC7B919FB49789DFB1EE6BB" bold="true" box="[1260,1273,1260,1279]" pageId="2" pageNumber="3">E</emphasis>
|
||
, which was annotated as flavonoids, while Chemogroup 2 (Aj-L, Ah-F, Af-F, and Ahv-F) was rich in tannins. On the other hand, diarylheptanoids were majorly represented in the remaining samples (Chemogroup 1). This pattern partially agreed with the plant parts-based distribution (
|
||
<figureCitation id="5EFC2A25FFC7B919FAEA792AFA62E72A" box="[1359,1413,1371,1390]" captionStart="Fig" captionStartId="3.[100,130,1281,1298]" captionTargetBox="[226,1361,154,1258]" captionTargetId="figure-514@3.[225,1362,152,1259]" captionTargetPageId="3" captionText="Fig. 2. The MS/MS spectral network of specialized metabolites contained in the bark, twigs, leaves, and fruits of A. japonica, A. firma, A. hirsuta, and A. hirsuta var. sibirica. Spectral nodes are colored according to the mean precursor ion intensity per different plant parts: bark, twigs, leaves, and fruits. Molecular families A–I are highlighted." figureDoi="http://doi.org/10.5281/zenodo.8294568" httpUri="https://zenodo.org/record/8294568/files/figure.png" pageId="2" pageNumber="3">Fig. 2</figureCitation>
|
||
), but it could be revealed that there are some exceptional cases such as Aj-F (rich in diarylheptanoids while the other fruits are abundant in tannins), Aj-L (rich in tannins while the other leaves are abundant in flavonoids), and Af-T and Af-B (rich in flavonoids while the other bark and twigs are abundant in diarylheptanoids). It is well known that plant specialized metabolite profiles vary based on a number of biotic as well as abiotic factors, such as diurnal changes, presence of herbivory or plant symbionts, nutrient availability and exposure to sunlight (
|
||
<bibRefCitation id="A2564B51FFC7B919FC9F7A27FBB4E42D" author="Bednarek, P. & Osbourn, A." box="[826,1107,1622,1641]" pageId="2" pageNumber="3" pagination="746 - 748" refId="ref7210" refString="Bednarek, P., Osbourn, A., 2009. Plant-microbe interactions: chemical diversity in plant defense. Science 324, 746 - 748. https: // doi. org / 10.1126 / science. 1171661." type="journal article" year="2009">Bednarek and Osbourn, 2009</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="A2564B51FFC7B919FBC57A27FB37E42D" author="Wink, M." box="[1120,1232,1622,1641]" pageId="2" pageNumber="3" pagination="1 - 19" refId="ref12336" refString="Wink, M., 2010. Introduction: biochemistry, physiology and ecological functions of secondary metabolites. In: Wink, M. (Ed.), Annual Plant Reviews Volume 40: Biochemistry of Plant Secondary Metabolism, second ed. Blackwell Publishing Ltd., pp. 1 - 19. https: // doi. org / 10.1002 / 9781444320503. ch 1." type="book chapter" year="2010">Wink, 2010</bibRefCitation>
|
||
). We were not able to explain the biological context of these exceptional localizations in
|
||
<taxonomicName id="01C74D23FFC7B919FA237A03FA28E4C1" box="[1414,1487,1650,1669]" class="Magnoliopsida" family="Betulaceae" genus="Alnus" kingdom="Plantae" order="Fagales" pageId="2" pageNumber="3" phylum="Tracheophyta" rank="species" species="firma">
|
||
<emphasis id="F4B3EAB2FFC7B919FA237A03FA28E4C1" bold="true" box="[1414,1487,1650,1669]" italics="true" pageId="2" pageNumber="3">A. firma</emphasis>
|
||
</taxonomicName>
|
||
and
|
||
<taxonomicName id="01C74D23FFC7B919FCF97AFFFC24E4E5" box="[860,963,1678,1697]" class="Magnoliopsida" family="Betulaceae" genus="Alnus" kingdom="Plantae" order="Fagales" pageId="2" pageNumber="3" phylum="Tracheophyta" rank="species" species="japonica">
|
||
<emphasis id="F4B3EAB2FFC7B919FCF97AFFFC24E4E5" bold="true" box="[860,963,1678,1697]" italics="true" pageId="2" pageNumber="3">A. japonica</emphasis>
|
||
</taxonomicName>
|
||
as metadata collected did not allow for a precise evaluation of contributing factors to the differences of the metabolic profiles observed here; however, differences in chemical profiles revealed here will provide a helpful guidance for further phytochemical studies on
|
||
<taxonomicName id="01C74D23FFC7B919FCF57A8FFC34E555" box="[848,979,1790,1809]" class="Magnoliopsida" family="Betulaceae" genus="Alnus" kingdom="Plantae" order="Fagales" pageId="2" pageNumber="3" phylum="Tracheophyta" rank="species" species="undetermined">
|
||
<emphasis id="F4B3EAB2FFC7B919FCF57A8FFC65E555" bold="true" box="[848,898,1790,1809]" italics="true" pageId="2" pageNumber="3">Alnus</emphasis>
|
||
species.
|
||
</taxonomicName>
|
||
</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |