This work was funded by the National Natural Science Foundation of China&#39;s Youth Program (No. 82304670).

1. Experimental preparation
<ol>
	<li>Make sure that during plant growth, the relative humidity of the greenhouse is 75%, the day/night temperatures are 20 &#176;C/10 &#176;C, the photoperiod is made up of 12 h day and 12 h night, and the light intensity is 100 &#181;mol&#183;m-2&#183;s-1. Provide irradiance via light-emitting diode (LED) lamps, keeping a distance of 30 cm between the LED lamp and the plant canopy. 
	NOTE: The photoperiod and light intensity are according to the number of sunshine hours during the growing period in Yunnan. Irradiance is routinely measured by a quantum sensor (see Table of Materials). Make the necessary changes in environmental parameters according to the medicinal plant&#39;s characteristics. Once these environmental conditions have been carefully calibrated based on the plant&#39;s characteristics and the regional sunlight profile, it is critical to maintain consistency in these settings throughout the duration of the experiment. Arbitrarily changes to the environmental parameters after their initial establishment could compromise the integrity of the experiment and the reliability of the results.</li>
	<li>Use a hydroponic approach18 to ensure the accuracy and specificity of our experiment. Make sure that the washed 2-year-old PPY seedlings (harvested from Wenshan, Yunnan Province (104&#176;11&#8242;E, 23&#176;04&#8242;N) are immersed in a 1/4 standard concentration of Hoagland&#39;s nutrient solution for 3 days for adapting.
	<ol>
		<li>Prepare the Hoagland&#39;s nutrient (see Table of Materials) solution according to the instructions; add 1.26 g of Hoagland powder and 0.945 g of Calcium nitrate powder into 4 L of purified water. Prepare the 13C6-Glucose (see Table of Materials) solution as described in the standard manual; add 0.2 g of 13C6-Glucose powder into 100 mL of purified water or Hoagland&#39;s nutrient solution.</li>
		<li>Prepare the hydroponic tank and oxygen pump (Figure 2; see Table of Materials). 
		NOTE: In the hydroponic setup, utilizing a 13C6-Glucose solution for labeling circumvents the influence of soil microorganisms, thereby ensuring that the solution directly contributes to the rapid synthesis of secondary metabolites in medicinal plants. To optimize the conditions for the absorption of 13C6-Glucose by the plants, it is imperative to regulate the flow rate of the oxygen pump between 4 and 8 L/min. This controlled flow rate is crucial to prevent the seedlings from floating atop the water surface, which could significantly hinder their ability to absorb the 13C6-Glucose effectively. Maintaining the seedlings submerged at the correct depth allows for efficient uptake of the labeling compound, thereby ensuring the success of the metabolite synthesis process in the hydroponic system.</li>
	</ol>
	</li>
</ol>
2. Carbon labeling experiment operation
<ol>
	<li>Set up four treatments and trace the synthesis organs and pathways of PS VII and PS II in PPY to demonstrate that leaves are the primary organ for synthesizing PS.
	<ol>
		<li>In Treatment 1, the PPY seedling is non-feeding; in Treatment 2, spray the PPY seedling leaves with 0.2% 13C6-Glucose; in Treatment 3, feed the PPY seedling rhizomes with 0.2% 13C6-Glucose; in Treatment 3, feed the PPY seedling at the stem incision with the 0.2% 13C6-Glucose (Figure 2A). 
		NOTE: Treatment 1 is set as a control group. Treatment 2 is set up to demonstrate that leaves can synthesize the PS. Treatments 3 and 4 are set up to demonstrate that rhizomes can take up 13C6-Glucose.</li>
		<li>In Treatments 1 and 2, culture the PPY seedlings in normal Hoagland&#39;s nutrient solution. Spray 5 mL of 0.2% 13C6-Glucose solution once in the morning, afternoon, and evening on the leaves of Treatment 2.</li>
		<li>In Treatments 3 and 4, culture the PPY seedlings in Hoagland&#39;s nutrient solution containing 0.2% 13C6-Glucose. In Treatment 4, use a scalpel (see Table of Materials) to cut off the stems of PPY seedlings from the middle but keep the vascular bundles attached.</li>
		<li>Allow each treatment to be labeled for 3 days, and change the nutrient solution every 1.5 days. Conduct the experiment using a randomized block design and repeat each treatment 3x. 
		NOTE: When spraying the leaves, it is important to place an absorbent paper behind the sprayed leaves. This prevents water droplets from falling into the nutrient solution below, which could otherwise affect the results of the experiment. The cuts in the stems are not all removed but have conduits attached that are visible to the naked eye.</li>
	</ol>
	</li>
</ol>
3. Sampling and preparation methods
<ol>
	<li>Collect leaves, stems, rhizomes, and roots from each treatment separately at the end of the 3-day 13C6-Glucose experiment. Wash the collected plant organs thoroughly to remove surface impurities. 
	NOTE: The absence of 13C abundance can be found by LC-MS analysis of the washing solution, which proves that the washing is clean19.</li>
	<li>Use an electric constant temperature blast drying oven (see Table of Materials) to dry the washed plant organs. Start at 105 &#176;C for 30 min and then set at 60 &#176;C until constant weight. Crush the dried plant organs into powder using a fully automatic sample rapid grinder (see Table of Materials). 
	NOTE: The powder is used for quantitative analysis of PS VII, II, and 13C/12C ratios in different plant organs.</li>
	<li>Use an electronic analytical balance (see Table of Materials) to accurately weigh 30 mg of powder for each sample and place it in 2 mL of 75% v/v ethanol-water. Use a CNC ultrasonic cleaner (see Table of Materials) and perform ultrasonic extraction at 25 &#176;C for 20 min (60 kHz), repeat 2x; merge the extraction solutions and prepare three parallel samples20. 
	NOTE: Errors in sample weight should be minimized.</li>
	<li>Use nitrogen to blow dry the extraction solution, then dissolve in chromatographic methanol to a volume of 1 mL. Before injection, use a 0.22 &#181;m organic phase filter (see Table of Materials) for filtration (Figure 2B). 
	NOTE: Do not blow out the extract when blow drying with nitrogen gas; the extract must be passed through a microporous membrane to ensure that impurities interfere minimally.</li>
</ol>
4. LC-MS setup and operation
<ol>
	<li>MS setting
	<ol>
		<li>Activate the vacuum pump by flipping its switch to the on position. Open the main valve on the argon gas cylinder, then adjust the partial pressure valve to achieve a target pressure of about 0.3 MPa. Following this, ensure the nitrogen gas valve is also opened.</li>
		<li>Launch the MS control software (see Table of Materials). Click on the heated electrospray ionization source (HESI Source) in the software panel, and enter the parameters, including the capillary voltage (3.0 kV for negative mode), the capillary temperature (350 &#176;C), sheath gas (N2) flow rate (35 arbitrary units), auxiliary gas (N2) flow rate (15 arbitrary units), through full scanning based on the molecular mass of PS (operated from m/z 100-1,500). Click on the Apply button to activate the ion source. 
		NOTE: Wait for at least 8 h to ensure a sufficient vacuum degree for the experimental conditions. Check that the gas pressure of argon and nitrogen is high enough before analysis.</li>
	</ol>
	</li>
	<li>LC prerun
	<ol>
		<li>Prepare mobile phase A by mixing 0.1% formic acid with water and use acetonitrile for mobile phase B (see Table of Materials). Degas both phases in an ultrasound bath sonicator for 15 minutes to eliminate dissolved gases. Then, connect mobile phase A to its respective fluid passage and do the same with mobile phase B. Finally, mix a 1:9 v/v methanol-water solution for the cleanout fluid and fill the pump and injector bottles manually. 
		NOTE: The frequency of the ultrasound bath sonicator is 40 kHz.</li>
	</ol>
	</li>
	<li>LC method establishment
	<ol>
		<li>Select the &#34;Instrument Setup&#34; button to access the method-editing window.</li>
		<li>Press the &#34;New&#34; button to initiate the creation of a new LC-MS instrument method.</li>
		<li>Set the total run time for the LC method. Then, input the necessary values for the pressure limit, total flow rate, flow gradient, sample temperature, column temperature, and ready temperature delta within the method-editing window. 
		NOTE: LC-MS instrument method settings are tailored to the analyte of interest and the type of liquid chromatography column utilized. For optimal results, use a flow rate of 0.3 mL/min with a BEH C18 column maintained at 40 &#176;C (see Table of Materials). The gradient elution is configured as follows: begin at 10% B, increasing linearly to 30% B over 8 min, then to 45% B from 8 to 16 min, to 50% B from 16 to 24 min, and to 70% B from 24 to 30 min. Immediately following this, revert the gradient to the initial 10% B condition within 0.1 min, holding it there for an additional 2.9 min. Inject a sample volume of 5 &#181;L for each analysis.LC-MS instrument method settings depend on the specific substance to be analyzed and the type of liquid chromatography column used21.</li>
	</ol>
	</li>
	<li>MS acquisition
	<ol>
		<li>In the Settings tab, choose either the General MS or MSn experiment type for the MS method configuration. Input the necessary values to set up the acquisition time, polarity (positive or negative), mass rangedivert valve number, and divert valve duration. After entering these details, click on the &#34;Save&#34; button to finalize and save these settings as an instrument method. 
		NOTE: The default settings without a chromatography column are as follows: acquisition time, 2 min; polarity, positive or negative; mass range, 100 to 1,500.</li>
	</ol>
	</li>
	<li>Perform multiple-stage mass spectrometry according to conventional methods.
	<ol>
		<li>Always employ data-dependent acquisition (DDA) mode for data collection in multi-stage mass spectrometry. In DDA mode, the mass spectrometer identifies the five most intense ions during each scan in the first stage of tandem MS, which are subsequently fragmented and examined in the second stage of tandem mass spectrometry. 
		NOTE: The collected MS and MS/MS data can be used for subsequent analysis and database searching.</li>
	</ol>
	</li>
</ol>
5. Manual data acquisition, analysis, and calculation
<ol>
	<li>Data acquisition and analysis
	<ol>
		<li>Double-click on the raw files to open all the mass spectra from MS. Manually calculate the m/z difference values between the ion and the corresponding fragment ions.</li>
		<li>Import the raw data files of the control and sample treatments and blanks into the mass spectrometry analysis software to identify PSs by accurately calculating the mass and the fragments of PS. 
		NOTE: PS VII and PS II are identified by comparison with retention times and coelution of authentic standard solutions. LC-MS detects a series of small peaks at the positions of the molecular ion peaks. The ratio of each series of isotopic ion peaks is constant. When exogenous 13C isotopes are given, the total 13C abundance in the plant increases, and the ratios of the series of isotope ion peaks of plant secondary metabolites change. The maximum tolerance of mass error is set at 5 ppm.</li>
		<li>Scan the mass range m/z 500-1,500 to identify the basic peak. Determine the accurate molecular weight to 4 decimal places, according to the precise molecular weight and retention time of the target molecule. Search for the peak area of serial isotope ion flow and derive values for M&#8722;, (M+1) &#8722;, (M+2) &#8722;, (M+3) &#8722;, and (M+4) &#8722;. When the mobile phase contains formic acid, the negative ions also include (M+COOH) &#8722;, (M+COOH+1) &#8722;, (M+COOH+2) &#8722;, (M+COOH+3) &#8722; and (M+COOH+4) &#8722;. 
		NOTE: Selecting peaks within the mass range m/z 500-1,500 improves the accuracy of identifying molecular ion peaks for PS VII and PS II. For instance, the negative ion mass spectrum of PS VII (C51H82O21, 1030.5349) mainly ionizes with COOH&#8722; ions in formic acid-water mixture. The observed serial isotope ions include 1029.53&#8722;, 1030.54&#8722;, 1031.54&#8722;, 1032.55&#8722;, 1033.55&#8722;, and extending to 1079.56&#8722;.</li>
	</ol>
	</li>
	<li>Data calculation
	<ol>
		<li>To reduce the effect of 13C natural abundance, calculate the ratios of labeled to unlabeled ion currents for each organ under different treatments, specifically focusing on the ratios (M+1)&#8722;/ M&#8722;, (M+2)&#8722;/M&#8722;, (M+3)&#8722;/ M&#8722; and (M+4)&#8722;/ M&#8722;. To differentiate between labeled and naturally occurring isotopes for an accurate assessment of 13C incorporation, calculate these ratios for n = 1, 2, 3, 4 using equation (1). 
		Ratio = A (M+2) &#8722;/ AM&#8722;&#160; &#160;(1) 
		PS: (M+2) &#8722; include (M+2+COOH) &#8722; and (M+2) &#8722;; M&#8722; include (M+COOH) - and M&#8722;. 
		NOTE: Here, M&#8722; refers to the combined peak areas of ion currents for (M+COOH) &#8722; and M &#8722;, primarily ionized with COOH&#8722; in formic acid-water. A represents the peak area. For example, the formulas to calculate PS VII ratios are illustrated as follows: 
		Ratio+1= (A1030.54+A1076.55)/(A1029.53+A1075.55) 
		Ratio+2= (A1031.54+A1077.55)/(A1029.53+A1075.55) 
		Ratio+3= (A1032.55+A1078.56)/(A1029.53+A1075.55) 
		Ratio+4= (A1033.55+A1079.56)/(A1029.53+A1075.55)</li>
	</ol>
	</li>
</ol>

Primary Organ Identification in Medicinal Plants Using 13C6&#45;Glucose Labeling

<ol>
	<li>Erb, M., Kliebenstein, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+secondary+metabolites+as+defenses,+regulators,+and+primary+metabolites:+The+blurred+functional+trichotomy.">Plant secondary metabolites as defenses, regulators, and primary metabolites: The blurred functional trichotomy.</a> Plant Physiol. 184 (1), 39-52 (2020).</li><li>Li, Y., Kong, D., Fu, Y., Sussman, M. R., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+developmental+and+environmental+factors+on+secondary+metabolites+in+medicinal+plants.">The effect of developmental and environmental factors on secondary metabolites in medicinal plants.</a> Plant Physiol Biochem. 148, 80-89 (2020).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+molecular+dissection+of+ginseng+(panax+ginseng+mey.)+germplasm+using+high-density+genic+snp+markers,+secondary+metabolites,+and+gene+expressions.">Genetic and molecular dissection of ginseng (panax ginseng mey.) germplasm using high-density genic snp markers, secondary metabolites, and gene expressions.</a> Front Plant Sci. 14, 1165349 (2023).</li><li>Chen, X., Wang, Y., Zhao, H., Fu, X., Fang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+dynamic+change+of+saponins+in+cyclocarya+paliurus+(batal.)+iljinskaja.">Localization and dynamic change of saponins in cyclocarya paliurus (batal.) iljinskaja.</a> PloS One. 14 (10), e0223421 (2019).</li><li>Yuan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+and+in+vivo+stable+isotope+labelling+of+central+carbon+metabolism+and+related+pathways+with+analysis+by+lc-ms/ms.">Ex vivo and in vivo stable isotope labelling of central carbon metabolism and related pathways with analysis by lc-ms/ms.</a> Nat Protoc. 14 (2), 313-330 (2019).</li><li>Cavalli, F. M. G., Bourgon, R., Vaquerizas, J. M., Luscombe, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specond:+A+method+to+detect+condition-specific+gene+expression.">Specond: A method to detect condition-specific gene expression.</a> Genome Biol. 12 (10), 101 (2011).</li><li>Epron, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulse-labelling+trees+to+study+carbon+allocation+dynamics:+A+review+of+methods,+current+knowledge+and+future+prospects.">Pulse-labelling trees to study carbon allocation dynamics: A review of methods, current knowledge and future prospects.</a> Tree Physiology. 32 (6), 776-798 (2012).</li><li>Varman, A. M., He, L., You, L., Hollinshead, W., Tang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+intrinsic+biosynthesis+yields+using+13c-based+metabolism+analysis.">Elucidation of intrinsic biosynthesis yields using 13c-based metabolism analysis.</a> Microb Cell Fact. 13 (1), 42 (2014).</li><li>Meng-Meng, G., Zhen-Yu, Z., Yu, Z., Qiu-Lin, Y., Ying, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+extraction+and+stable+isotope+determination+of+different+biomarkers+from+single+leaf+of+reticulate+vein+plants.">Systematic extraction and stable isotope determination of different biomarkers from single leaf of reticulate vein plants.</a> Journal of Shaanxi University of Science &amp; Technology. 41 (01), 72-79 (2023).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+convenient+lc-ms+method+for+assessment+of+glucose+kinetics+in+vivo+with+d-[13c6]glucose+as+a+tracer.">A convenient lc-ms method for assessment of glucose kinetics in vivo with d-[13c6]glucose as a tracer.</a> Clin Chem. 55 (3), 527-532 (2009).</li><li>Chassy, A. W., Adams, D. O., Waterhouse, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+phenolic+metabolism+in+vitis+vinifera+berries+with+13c6-phenylalanine:+Implication+of+an+unidentified+intermediate+reservoir.">Tracing phenolic metabolism in vitis vinifera berries with 13c6-phenylalanine: Implication of an unidentified intermediate reservoir.</a> J Agric Food Chem. 62 (11), 2321-2326 (2014).</li><li>Lozac'h, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cams+for+14c+microtracer+adme+studies:+Opportunities+to+change+the+current+drug+development+paradigm.">Evaluation of cams for 14c microtracer adme studies: Opportunities to change the current drug development paradigm.</a> Bioanalysis. 10 (5), 321-339 (2018).</li><li>Sulyok, M., Stadler, D., Steiner, D., Krska, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+an+lc-ms/ms-based+dilute-and-shoot+approach+for+the+quantification+of+&amp;gt;+500+mycotoxins+and+other+secondary+metabolites+in+food+crops:+Challenges+and+solutions.">Validation of an lc-ms/ms-based dilute-and-shoot approach for the quantification of &amp;gt; 500 mycotoxins and other secondary metabolites in food crops: Challenges and solutions.</a> Anal Bioanal Chem. 412 (11), 2607-2620 (2020).</li><li>Serra, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter-laboratory+comparison+of+elemental+analysis+and+gas+chromatography+combustion+isotope+ratio+mass+spectrometry+(gc-c-irms).+Part+i:+Delta13c+measurements+of+selected+compounds+for+the+development+of+an+isotopic+grob-test.">Inter-laboratory comparison of elemental analysis and gas chromatography combustion isotope ratio mass spectrometry (gc-c-irms). Part i: Delta13c measurements of selected compounds for the development of an isotopic grob-test.</a> J Mass Spectrom. 42 (3), 361-369 (2007).</li><li>Jung, J. -. Y., Oh, M. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotope+labeling+pattern+study+of+central+carbon+metabolites+using+gc/ms.">Isotope labeling pattern study of central carbon metabolites using gc/ms.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 974, 101-108 (2015).</li><li>Ding, Y. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+traditional+uses,+phytochemistry,+and+pharmacological+properties+of+paris+l.+(liliaceae):+A+review.">The traditional uses, phytochemistry, and pharmacological properties of paris l. (liliaceae): A review.</a> J Ethnopharmacol. 278, 114293 (2021).</li><li>Cunningham, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paris+in+the+spring:+A+review+of+the+trade,+conservation+and+opportunities+in+the+shift+from+wild+harvest+to+cultivation+of+paris+polyphylla+(trilliaceae).">Paris in the spring: A review of the trade, conservation and opportunities in the shift from wild harvest to cultivation of paris polyphylla (trilliaceae).</a> J Ethnopharmacol. 222, 208-216 (2018).</li><li>Madikizela, L. M., Ncube, S., Chimuka, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+of+pharmaceuticals+by+plants+grown+under+hydroponic+conditions+and+natural+occurring+plant+species:+A+review.">Uptake of pharmaceuticals by plants grown under hydroponic conditions and natural occurring plant species: A review.</a> Sci Total Environ. 636, 477-486 (2018).</li><li>Tagami, K., Uchida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Online+stable+carbon+isotope+ratio+measurement+in+formic+acid,+acetic+acid,+methanol+and+ethanol+in+water+by+high+performance+liquid+chromatography-isotope+ratio+mass+spectrometry.">Online stable carbon isotope ratio measurement in formic acid, acetic acid, methanol and ethanol in water by high performance liquid chromatography-isotope ratio mass spectrometry.</a> Anal Chim Acta. 614 (2), 165-172 (2008).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combination+of+red+and+blue+light+increases+the+biomass+and+steroidal+saponin+contents+of+paris+polyphylla+var.+Yunnanensis.">The combination of red and blue light increases the biomass and steroidal saponin contents of paris polyphylla var. Yunnanensis.</a> Ind Crops Prod. 194, 116311 (2023).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+distribution,+metabolism+and+absorption+of+rhizoma+paridis+saponins+in+the+rats.">Tissue distribution, metabolism and absorption of rhizoma paridis saponins in the rats.</a> J Ethnopharmacoly. 273, 114038 (2021).</li><li>Peng, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+study+of+differences+in+the+types+and+contents+of+steroidal+saponins+in+paris.+Polyphylla+smith+var.+Chinensis+(franch.)+hara.">Progress in the study of differences in the types and contents of steroidal saponins in paris. Polyphylla smith var. Chinensis (franch.) hara.</a> Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology. 24 (05), 2014-2025 (2022).</li><li>Alami, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+developments+in+medicinal+plant+genomics+enabled+the+diversification+of+secondary+metabolites'+biosynthesis.">The current developments in medicinal plant genomics enabled the diversification of secondary metabolites' biosynthesis.</a> Int J Mol Sci. 23 (24), 15932 (2022).</li><li>Wen, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthesis+of+paris+saponin+vii+mainly+occurs+in+leaves+and+is+promoted+by+light+intensity.">The synthesis of paris saponin vii mainly occurs in leaves and is promoted by light intensity.</a> Front Plant Sci. 14, 1199215 (2023).</li><li>Siadjeu, C., Pucker, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medicinal+plant+genomics.">Medicinal plant genomics.</a> BMC Genomics. 24 (1), 429 (2023).</li><li>Tian, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+phenomics+of+arabidopsis+thaliana:+Metabolic+profiling+by+one-+and+two-dimensional+nuclear+magnetic+resonance+spectroscopy+and+transcriptome+analysis+of+albino+mutants.">Top-down phenomics of arabidopsis thaliana: Metabolic profiling by one- and two-dimensional nuclear magnetic resonance spectroscopy and transcriptome analysis of albino mutants.</a> J Biol Chem. 282 (25), 18532-18541 (2007).</li><li>Masakapalli, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+flux+phenotype+of+tobacco+hairy+roots+engineered+for+increased+geraniol+production.">Metabolic flux phenotype of tobacco hairy roots engineered for increased geraniol production.</a> Phytochemistry. 99, 73-85 (2014).</li><li>Schwender, J., Ohlrogge, J. B., Shachar-Hill, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flux+model+of+glycolysis+and+the+oxidative+pentosephosphate+pathway+in+developing+brassica+napus+embryos.">A flux model of glycolysis and the oxidative pentosephosphate pathway in developing brassica napus embryos.</a> J Biol Chem. 278 (32), 29442-29453 (2003).</li></ol>

The authors declare no competing financial interests.

The successful implementation of this protocol hinges on comprehensive research into plant physiological properties, tissues, organs, and secondary metabolites. The experimental design approach outlined in the protocol lays a robust foundation for investigating the biosynthetic pathways of plant secondary metabolites. The critical factors in this experiment are (1) determining the age of the perennial seedlings and (2) choosing the correct isotope labeling-detection timing. The medicinal plants are categorized into peren...

The biosynthetic pathways of secondary metabolites in plants are intricate and diverse, involving highly specific and diverse accumulation organs1. At present, the specific synthesis sites and responsible organs for secondary metabolites in many medicinal plants are not well-defined. This ambiguity poses a significant obstacle to the strategic advancement and implementation of cultivation methods designed to optimize both the yield and quality of medicinal materials.
Molecular biology, biochemical, and isotope labeling techniques are extensively employed to unravel the synthesis pathways and sites of secondary metabolites in medicinal plants2,3,4,5, and each of these methodologies exhibits unique strengths and limitations, such as differences in efficiency and accuracy. Molecular biology approaches, for instance, offer high precision in pinpointing the sites within biosynthetic pathways but are notably time-intensive. Their utility is further constrained for species lacking publicly available genomic sequences, rendering these techniques less viable for such cases6. In contrast, isotope labeling techniques, employing isotopic ratios like 3C/12C, 2H/1H, and 18O/16O, provide a rapid and accessible means to investigate the synthesis, transport, and storage mechanisms of secondary metabolites7,8. They can reveal the spatial distribution of organic compounds and stable isotopes in leaves, thereby allowing the reconstruction of environmental conditions experienced by the leaves throughout their life cycle9. Furthermore, the application of external isotopic labels, such as 13C6-Glucose10 and 13C6-Phenylalanine11, enables the generation of carbon-labeled secondary metabolites, enhancing our understanding of their production and function.
Traditional carbon isotope labeling techniques encounter challenges in pinpointing the specific organs responsible for the synthesis of secondary metabolites due to the highly species-specific nature of their biosynthetic pathways and transport mechanisms. Liquid chromatography-mass spectrometry (LC-MS) has risen to prominence as a pivotal analytical instrument in this arena, offering a robust method for tracking exogenous isotopes in the chemical synthesis of drugs and investigating in vivo processes such as absorption, distribution, metabolism, and excretion12. The superior sensitivity, straightforwardness, and reliability of LC-MS make it an ideal choice for monitoring the production of secondary metabolites in plants13. In recent times, LC-MS has become increasingly favored for its application in external isotope labeling techniques, which enables the evaluation of labeling efficiency across different samples. This methodology provides critical insights into the primary organs engaged in the synthesis of secondary metabolites in medicinal plants, serving as an invaluable complement to biological methods for identifying the synthesis organs of these compounds14,15. Consequently, this approach not only facilitates the comparison of labeling efficiencies among various specimens but also sheds light on the key organs implicated in the generation of plant secondary metabolites, thereby enhancing our understanding of their biosynthesis.
We introduced a novel method that combines carbon isotope labeling with LC-MS detection to identify the primary organs responsible for synthesizing secondary metabolites in medicinal plants. Paris saponin (PS) has a variety of pharmacological activities such as anticancer, immunomodulation, and anti-inflammation16, and PPY is in increasing demand17. Therefore, we used PPY seedlings as research subjects and deciphered that leaves are the primary organ to synthesize the Paris saponin VII (PS VII) (Figure 1B) by using the 13C6-Glucose labeling associated with the LC-MS method. Our approach included four different treatments involving 13C6-Glucose feeding to leaf, rhizome, and stem-vascular bundles, as well as a non-feeding control. The choice of 13C6-Glucose is strategic, as it is swiftly metabolized into acetyl coenzyme A via respiration, which then facilitates PS synthesis. Employing the natural abundance of 13C, we utilized a Gas Chromatography-Stable Isotope Ratio Mass Spectrometer (GC-IRMS) system to assess the 13C/12C ratios across various plant organs and to analyze the isotopic ion peak ratios in PS VII and Paris saponins II (PS II) (Figure 1B) molecules. Our methodology, which leverages 13C-labeled plant secondary metabolite precursors and cutting-edge mass spectrometry techniques, offers a simpler and more accurate alternative to conventional carbon isotope labeling methods. This novel approach not only deepens our comprehension of the organs involved in secondary metabolite synthesis in medicinal plants but also lays a solid groundwork for future explorations into the biosynthetic pathways of these compounds.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 % Formic acid water</td>
			<td>Chengdu Kelong Chemical Reagent Factory</td>
			<td>44890</td>
			<td></td>
		</tr>
		<tr>
			<td>13C6-Glucose powder</td>
			<td>MERCK</td>
			<td>110187-42-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Chengdu Kelong Chemical Reagent Factory</td>
			<td>44890</td>
			<td></td>
		</tr>
		<tr>
			<td>AUTOSAMPLER VIALS</td>
			<td>Biosharp Biotechnology Company</td>
			<td>44866</td>
			<td></td>
		</tr>
		<tr>
			<td>BEH C18 column</td>
			<td>Waters,Milfor,MA</td>
			<td>1.7&#956;m,2.1*100 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>CNC ultrasonic cleaner</td>
			<td>Kunshan Ultrasound Instrument Co., Ltd</td>
			<td>KQ-600DE</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound DiscovererTM&#160; software</td>
			<td>Thermo Scientific, Fremont,CA</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound DiscovererTM&#160; software&#160;</td>
			<td>Thermo Scientific,Fremont,CA</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric constant temperature blast drying oven</td>
			<td></td>
			<td>DHG-9146A</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic analytical balance</td>
			<td>Sedolis Scientific Instruments Beijing Co., Ltd</td>
			<td>SOP</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Chengdu Kelong Chemical Reagent Factory</td>
			<td>44955</td>
			<td></td>
		</tr>
		<tr>
			<td>Fully automatic sample rapid grinder</td>
			<td>Shanghai Jingxin Technology</td>
			<td>Tissuelyser-48</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Chromatography-Stable Isotope Ratio Mass Spectrometer</td>
			<td>Thermo Fisher</td>
			<td>Delta V Advantage</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoagland solution</td>
			<td>Sigma-Aldrich</td>
			<td>H2295-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroponic tank</td>
			<td>JRD</td>
			<td>1020421</td>
			<td></td>
		</tr>
		<tr>
			<td>Isodat software</td>
			<td>Thermo Fisher Scientific</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid chromatography high-resolution mass spectrometry</td>
			<td>Agilent Technology&#160;</td>
			<td>Agilent 1260 -6120&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen manufacturing instrument</td>
			<td>PEAK SCIENTIFIC</td>
			<td>Genius SQ 24</td>
			<td></td>
		</tr>
		<tr>
			<td>Organic phase filter</td>
			<td>Tianjin Jinteng Experimental Equipment Co., Ltd</td>
			<td>44890</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen pump</td>
			<td>Magic Dragon</td>
			<td>MFL</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum sensor</td>
			<td>Highpoint</td>
			<td>UPRtek</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Handskit</td>
			<td>11-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprinkling can</td>
			<td>CHUSHI</td>
			<td>WJ-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Xcalibur&#160; software</td>
			<td>Thermo Fisher Scientific</td>
			<td>4.2</td>
			<td></td>
		</tr>
	</tbody>
</table>

13c6&#45;glucose labeling associated with lc&#45;ms&#58; identification of plant primary organs in secondary metabolite synthesis

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary metabolite in Parispolyphylla var. yunnanensis (Franch.) Hand. -Mzt. (PPY), Paris saponin (PS) has a variety of pharmacological activities and PPY is in increasing demand. This study established leaf, rhizome, and stem-vascular-bundle 13C6-Glucose feeding and non-feeding four treatments to precisely certify the primary organs involved in Paris saponins VII (PS VII) synthesis. By combining liquid chromatography-mass spectrometry (LC-MS), the 13C/12C ratios of leaf, rhizome, stem, and root in different treatments were quickly and accurately calculated, and four types of PS isotopic ion peak(M&#8722;) ratios were found: (M+1) &#8722;/M&#8722;, (M+2) &#8722;/M&#8722;, (M+3) &#8722;/M&#8722; and (M+4) &#8722;/M&#8722;. The results showed that the ratio of 13C/12C in the rhizomes of the stem-vascular-bundle and rhizome feeding treatments was significantly higher than that in the non-feeding treatment. Compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves increased significantly under leaf and stem-vascular-bundle feeding treatments. Simultaneously, compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves under rhizome treatment showed no significant difference. Furthermore, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the stem, root, and rhizome showed no differences among the four treatments. Compared to the non-feeding treatment, the ratio of the Paris saponin II (PS II) molecule (M+2) &#8722;/M&#8722; in leaves under leaf feeding treatment showed no significant difference, and the (M+3) &#8722;/M&#8722; ratio of PS II molecules in leaves under leaf feeding treatment were lower. The data confirmed that the primary organ for the synthesizing of PS VII is the leaves. It lays the foundation for future identification of the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants.

To confirm that 13C6-Glucose supply in rhizomes was successful, we further analyzed the 13C/12C isotope ratios in rhizomes. The 13C /12C isotope ratios of Treatments 3 and 4 were much higher than those of Treatment 2 (Figure 1A). The results indicated that 13C6-Glucose from Treatment 3 and 4 entered the rhizomes through ingestion.
The ratios of 13C isotope peaks, suc...

Read this Scientific Article Protocol about Author Spotlight: Plant Primary Organs Profiling Using 13C6-Glucose Labeling and LC-MS at JoVE.com

Author Spotlight: Plant Primary Organs Profiling Using 13C6&#45;Glucose Labeling and LC&#45;MS

The developed method of 13C6-Glucose labeling combined with liquid chromatography high-resolution mass spectrometry is versatile and lays the foundation for future studies on the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants, as well as the comprehensive utilization of these secondary metabolites.

13C6&#45;Glucose Labeling Associated with LC&#45;MS&#58; Identification of Plant Primary Organs in Secondary Metabolite Synthesis

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary ...

13c6-glucose-labeling-associated-with-lc-ms-identification-plant

Research

JoVE Journal

Chemistry

504 Views.  Chengdu University of Traditional Chinese Medicine. The developed method of 13C6-Glucose labeling combined with liquid chromatography high-resolution mass spectrometry is versatile and lays the foundation for future studies on the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants, as well as the comprehensive utilization of these secondary metabolites.

Video: Author Spotlight: Plant Primary Organs Profiling Using 13C6-Glucose Labeling and LC-MS

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) membranes have been under development since the 1990's and have become a common method to template the synthesis of high aspect ratio nanostructures, mostly by electrochemical growth or pore-wetting. Recently, these membranes have become commercially available in a wide range of pore sizes and densities, leading to an extensive library of functional nanostructures being synthesized from AAO membranes. These include composite nanorods, nanowires and nanotubes made of metals, inorganic materials or polymers 3-10. Nanoporous membranes have been used to synthesize nanoparticle and nanotube arrays that perform well as refractive index sensors, plasmonic biosensors, or surface enhanced Raman spectroscopy (SERS) substrates 11-16, as well as a wide range of other fields such as photo-thermal heating 17, permselective transport 18, 19, catalysis 20, microfluidics 21, and electrochemical sensing 22, 23. Here, we report a novel procedure to prepare gold nanotubes in AAO membranes. Hollow nanostructures have potential application in plasmonic and SERS sensing, and we anticipate these gold nanotubes will allow for high sensitivity and strong plasmon signals, arising from decreased material dampening 15.

Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Synthesis of Plant Phenol-derived Polymeric Dyes for Direct or Mordant-based Hair Dyeing

Effective hair dyeing through in situ incubation of keratin hair with the products of fungal laccase-catalyzed polymerization of plant phenols has been previously demonstrated. However, the dyeing process takes a long time to complete compared to commercial hair-dyeing products. To overcome this bottleneck, pre-synthesized polymeric products of the oxidative reaction of Trametes versicolor laccase on catechin and catechol, either with or without mordant agents (e.g., FeSO4), were here employed to achieve permanent keratin hair dyeing in various colors and shades. The laccase action in acidic sodium acetate buffer led to a deep black coloration after coupling reactions between the plant phenols. The colored dye products were then desalted and concentrated with ultrafiltration. The dyes, with or without mordant agents, caused a significant increase in &#916;E values (i.e., color difference value) in gray human hair within 2.5 hours. In addition, different keratin colors and shades were induced depending upon the mordanting and pH changes. The dyed hair also exhibited a strong resistance to detergent treatments, indicating that our methods can give rise to permanent hair dyeing.&#160;Overall, our work has provided novel insight into developing eco-friendly hair-dyeing methods as alternatives to commercial toxic diamine-based dyes.

Here, we present a protocol to use pre-synthesized polymeric products derived from fungal laccase-catalyzed polymerization of plant phenols, either with or without mordant agents (e.g., FeSO4), to induce detergent-resistant keratin hair dyeing within 2.5 hours.

Effective hair dyeing through in situ incubation of keratin hair with the products of fungal laccase-catalyzed polymerization of plant phenols has been ...

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital structural and protective role in the development and life of higher plants. Although the intricate mechanisms regulating lignification processes in vivo strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher them. In a simple three-step workflow, the dual labeling protocol presented herein enables bioimaging studies of actively lignifying zones of plant tissues. The first step consists in the metabolic incorporation of two independent chemical reporters, surrogates of the two native monolignols that give rise to lignin H- and G-units. After incorporation into growing lignin polymers, each reporter is then specifically labeled with its own fluorescent probe via a sequential combination of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin autofluorescence, this approach leads to the generation of three-color localization maps of lignin within plant cell walls by confocal fluorescence microscopy and provides precise spatial information on the presence or absence of active lignification machinery at the scale of plant tissues, cells and different cell wall layers.

BLISS, a dual labeling protocol for studying lignification dynamics, was developed. Using synthetic monolignol reporters and a sequential combination of SPAAC and CuAAC bioorthogonal click reactions, this methodology paves the way to in-depth analysis of the factors that regulate the biogenesis of lignins in planta.

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...