Integrative Approaches for the Identification and Localization of … · Integrative Approaches for the Identiﬁcation and Localization of Specialized Metabolites in Tripterygium

Integrative Approaches for the Identification andLocalization of Specialized Metabolites inTripterygium Roots1[OPEN]

B. Markus Lange*, Justin T. Fischedick, Malte F. Lange2, Narayanan Srividya, Dunja Šamec, andBrenton C. Poirier

Institute of Biological Chemistry and M.J. Murdock Metabolomics Laboratory, Washington State University,Pullman, Washington 99164–6340 (B.M.L., J.T.F., N.S., D.Š., B.C.P.); Undergraduate Program in Biochemistry,University of Washington, Seattle, Washington 98195–7350 (M.F.L.); and RuCer Boškovi�c Institute, Departmentof Molecular Biology, HR–10002 Zagreb, Croatia (D.Š.)

ORCID IDs: 0000-0001-6565-9584 (B.M.L.); 0000-0001-7934-7987 (N.S.); 0000-0002-0600-6847 (D.Š.).

Members of the genus Tripterygium are known to contain an astonishing diversity of specialized metabolites. The lack of authenticstandards has been an impediment to the rapid identification of such metabolites in extracts. We employed an approach thatinvolves the searching of multiple, complementary chromatographic and spectroscopic data sets against the Spektraris databaseto speed up the metabolite identification process. Mass spectrometry-based imaging indicated a differential localization oftriterpenoids to the periderm and sesquiterpene alkaloids to the cortex layer of Tripterygium roots. We further provide evidencethat triterpenoids are accumulated to high levels in cells that contain suberized cell walls, which might indicate a mechanism forstorage. To our knowledge, our data provide first insights into the cell type specificity of metabolite accumulation in Tripterygiumand set the stage for furthering our understanding of the biological implications of specialized metabolites in this genus.

Extracts from plants of the genus Tripterygium, mostprominently Tripterygium wilfordii (Celastraceae; alsoknown as léi gōng téng in Mandarin, which can betranslated to thunder god vine), have a long history intraditional Chinese medicine as a remedy for diverseailments ranging from sores to fever and inflammation(first being mentioned in herbal compendia of the15th century; Helmstädter, 2013). Root extracts werelater evaluated by allopathic medicine, but several

randomized controlled clinical trials (conducted duringthe 1980s to 2000s) that evaluated the efficacy for treatingrheumatoid arthritis reached inconsistent conclusions (Liuet al., 2013). Challenges for standardization remain be-cause a plethora of metabolites have been isolated andstructurally characterized from Tripterygium extracts(many ofwhich are bioactive), including sesquiterpenoids,diterpenoids, triterpenoids, and sesquiterpene pyridinealkaloids (Brinker et al., 2007). Themost promising clinicalcandidate is a derivative of the diterpene triepoxide,triptolide, termed F60008. A phase I study with thiscompound in France was only partially successful dueto the toxicity of the semisynthetic derivative (Kitzenet al., 2009). There is still considerable enthusiasm forminnelide (Arora et al., 2015), a prodrug derivative oftriptolide, which is currently undergoing phase I clinicaltrials as a treatment for advanced gastrointestinal tu-mors in the United States (clinicaltrials.gov identifierNCT01927965). Other metabolites with encouragingpreclinical performance are the appetite-reducing tri-terpenoid quinone celastrol (Liu et al., 2015) and theimmunosuppressive sesquiterpene pyridine alkaloidwilfordine (and naturally occurring structural analogs;Duan et al., 1999).

While the contents of bioactive specialized metabo-lites in commercial Tripterygium root extracts vary sig-nificantly between growing areas in China, triptolideis always present in very low concentrations (less than0.008% of dry biomass), sesquiterpene pyridine alka-loids range between 0.0001% and 0.1% of dry biomass,and celastrol can accumulate up to about 1% of dry

1 This work was supported by the National Institutes of Health(grant no. RC2GM092561 to B.M.L.) and by McIntire-Stennis formulafunds from the Agricultural Research Center at Washington StateUniversity (to B.M.L.).

2 Present address: Gordon Center for Integrative Sciences, Univer-sity of Chicago, Chicago, IL 60637.

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is: B.Markus Lange ([email protected]).

B.M.L. conceived of the project, designed the experiments, andperformed chromatography on Tripterygium extracts; J.T.F. estab-lished the methods for metabolite quantitation and imaging; M.F.L.generated Spektraris database records based on literature searches;N.S. performed NMR experiments and interpreted the data; D.Š.performed metabolite imaging experiments and processed the data;B.C.P. performed microscopy with roots and measured the phyto-sterol content; B.M.L. wrote the article with contributions from allauthors.

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biomass (Zeng et al., 2013; Zhuo et al., 2013; Guo et al.,2014). To improve the yields of active principles ex-tractable from Tripterygium roots, an understanding ofthe factors that control their accumulation is highlydesirable. Specializedmetabolites often are found onlyin certain plant lineages and are accumulated in spe-cialized anatomical structures and cell types (Lange,2015). The spatially restricted occurrence of thesemetabolites presents challenges for analytical chem-istry. When bulk organs or tissues are processed toobtain sufficient material for metabolite quantitation,information about the localization of specialized me-tabolites is lost.Newer technologies using mass spectrometry-based

imaging (MSI) have been developed to allow the de-tection and mapping of metabolites at cellular resolu-tion (Bjarnholt et al., 2014). One approach involves laserdesorption ionization with or without prior applicationof a chemical matrix to aid with ionization (commonlyused acronyms are MALDI [for matrix-assisted laser-desorption ionization] and LDI, respectively). Thesample is introduced into a loading chamber and israstered with respect to a laser as stationary ionizationsource, whilemass spectra are recorded at each positionacross the sample. The signal intensity for an ion canthen be plotted versus the XY position on the specimen,thereby enabling the generation of an ion map or massspectral image. While the chemical diversity and theoccurrence of several cell types in close proximity havecomplicated the development of robust LDI- andMALDI-MSImethodswith plant sections, exciting recent successesinclude the imaging of various lipid species in cotton(Gossypium hirsutum) seed embryos (Horn et al., 2012), thelocalization of defense phenalenone-type phytoalexins inlesions caused by nematode infection of banana (Musaspp.) roots (Hölscher et al., 2014), and the differential ac-cumulation of secoisolariciresinol diglucoside (lignan) andcyanogenic glycosides in flax (Linum usitatissimum) seeds(Dalisay et al., 2015).The high-confidence identification of metabolites in

highly complex plant matrices is still a challenge. Whileseveral online mass spectral databases now allowspectral comparisons for thousands of metabolites, in-cluding those occurring uniquely in plants, the cover-age of open-source search tools for NMR spectra ismuch narrower (for review, see Lange, 2016). We de-veloped Spektraris as a one-stop source for the inte-grative analysis of both accurate mass and NMR data(Cuthbertson et al., 2013; Fischedick et al., 2015). Aspart of this study, we updated the Spektraris databasewith multiple spectral data types for metabolites oc-curring in the genus Tripterygium. We then fractionatedroot extracts, obtained chromatographic and spectro-scopic data sets for these fractions, and identified themajor constituents by searching against Spektraris,thereby demonstrating the utility of the comprehensiveonline spectral resource. MALDI-MSI indicated a dif-ferential localization of quinone methide triterpenoidsto the periderm and of sesquiterpene pyridine alkaloidsto the cortex layer of Tripterygium roots, which provides

important cellular context for follow-up studies to as-sess the biological functions of these classes of struc-turally complex metabolites.

RESULTS

Quantitation of Specialized Metabolites in TripterygiumRoot Extracts

Before attempting to identify and assess the localiza-tion of specialized metabolites in roots of Tripterygiumregelii, a robust method for the quantitation of knownactive principles had to be validated. Homogenates fromroot samples were extracted with acetone, and metabo-lites were separated and detected, based on previouslypublished protocols, using HPLC-quadrupole time offlight (QTOF)-mass spectrometry (MS; Cuthbertsonet al., 2013; Liu et al., 2013; Fischedick et al., 2015). Au-thentic standards of triptolide (Fig. 1A) and celastrol(Fig. 1B) were readily available and therefore served todetermine the reproducibility, sensitivity, ion suppres-sion, and extraction efficiency of the protocol. When theelectrospray ionization source was operated in positivemode, the highest triptolide peak intensities wereobtained for [M+H]+, with [M+Na]+, and [M+K]+ ionsoccurring at significantly lower abundances (Fig. 1, Cand D). In addition to relative retention time and mo-lecular ion (accurate mass time [AMT] tag; Cuthbertsonet al., 2013), tandem mass spectrometry (MS/MS) frag-mentation patterns were used as a criterion for peakidentification (Fig. 1, E and F). The quantitation of trip-tolide was performed by recording peak intensity inextracted ion chromatograms for [M+H]+. Celastrol alsowas identified based on comparisons of AMT tag andMS/MS data (Fig. 1, G, H, K, and L), which matched therecords deposited in the Spektraris and MassBank da-tabases (Horai et al., 2010; Cuthbertson et al., 2013). Thediode array detector (DAD) trace at 424 nmwas used forthe quantitation of celastrol (Fig. 1, I and J). Extractionefficiency (recovery), reproducibility of the extraction,linearity of detection, limits of detection and quantita-tion, and matrix effects were evaluated and a highlyrobust method was validated (for details, see “Materialsand Methods” and Supplemental Table S1).

Using thismethod, the contents of triptolide and celastrolin T. regelii roots were determined as 11.8 mg g21 and12.4mgg21, respectively (Fig. 2A). These concentrations arein the same range as those reported by others (toward thelower end for triptolide and among the highest for celastrol;Zeng et al., 2013; Zhuo et al., 2013; Guo et al., 2014). Trip-tolide alsowasdetected in extracts of stems (5.2mgg21) andleaves (6.4 mg g21), whereas only trace amounts (below thelimit of quantitation [LOQ]) of celastrol were detectable inthese organs (Fig. 2A). Visual inspection of T. regelii rootsrevealed three main morphological types: white lateralroots (youngest root tissue), orange roots (mediumage roottissue), andwoody roots (oldest root tissue; Fig. 2B).White,orange, and woody root material was collected separately,and metabolites were extracted and analyzed by HPLC-QTOF-MS. Triptolide was most abundant in woody roots

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Specialized Metabolite Localization in Tripterygium

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(16.5 mg g21), with lesser concentrations in orange roots(9.8 mg g21), and the lowest abundance in lateral roots(0.6 mg g21; Fig. 2A). Celastrol abundance was highest inorange roots 17.8 mg g21), with lower quantities in woodyroots (8.6mg g21) and lateral roots (6.6mg g21; Fig. 2A). Toourknowledge, this is thefirst study to report onmetaboliteconcentrations in different root types ofTripterygiumplants;therefore, we do not have a reference for comparison.

Development of Spectral Libraries to Expedite theIdentification of Specialized Metabolites inTripterygium Extracts

Very few standards for specialized metabolites occur-ring in Tripterygium extracts are commercially available,

which is a significant impediment to the annotation ofpeaks in HPLC-QTOF-MS runs at high confidence. Asa first step to remedy this situation we assembled thecurrently available information on the physicochemicalproperties of Tripterygium metabolites and integratedthese data sets into the Spektraris online database(Cuthbertson et al., 2013; Fischedick et al., 2015). Basedon our literature searches (as of June 2016), 415 spe-cialized metabolites had been isolated and character-ized from Tripterygium. Of these metabolites, 105 areabietane diterpenes, 22 are kaurane diterpenes, 86 aredehydroagarofuran sesquiterpenes, 64 are sesquiter-pene alkaloids, three are spermidine alkaloids, 26 arefriedelane triterpenes, 38 are friedooleanane triterpenes,48 are oleanane triterpenes, 15 are ursane triterpenes,

Figure 1. A and B, Identification of triptolide (A) and celastrol (B) in Tripterygium root extracts. C and D, HPLC-QTOF-MS de-tection (atm/z 361.1648 in positive mode) of a triptolide standard (C) and the corresponding root metabolite (D). E and F, MS/MSfragmentation obtained at 30 eV with a triptolide standard (E) and the corresponding root metabolite (F). G and H, HPLC-QTOF-MS detection (at m/z 451.2844 in positive mode) of a celastrol standard (G) and the corresponding root metabolite (H). I and J,HPLC-DAD detection at 424 nm of a celastrol standard (I) and the corresponding root metabolite (J). K and L, MS/MS frag-mentation obtained at 30 eV with a celastrol standard (K) and the corresponding root metabolite (L).

458 Plant Physiol. Vol. 173, 2017

Lange et al.



and eight additional metabolites belong to other struc-tural classes (Supplemental Table S2). Spectral data forthe most commonly identified metabolites from rootextracts (a total of 115) were integrated into Spektraris(Fig. 3). Following the recent integration of selected datasets from the NAPROC-13 database (López-Pérez et al.,2007), the Spektraris database now contains NMR rec-ords for over 20,000 plant natural products (http://langelabtools.wsu.edu/nmr).Acetone extracts of T. regelii were then analyzed by

HPLC-QTOF-MS and spectral data (exact mass and cal-culated elemental formula) compared with the updatedSpektraris database. However, the annotation of peakswas still not satisfactory due to the frequent occurrence ofisobars (metabolites with the samemonoisotopic mass) inTripterygium; therefore, we proceeded with additionalcharacterizations. NMR spectroscopy can provide unam-biguous data for the identification of isomers, but, forcomplex structures, highly purified fractions containinglarger quantities are generally required (even with mod-ern cryoprobes). We wanted to investigate if the spectralsearch approach enabled by the Spektraris databasewould yield peak annotations with only partially purifiedfractions. Since our greenhouse-grown materials werelimited, we obtained T. wilfordii roots from a commercialsupplier in China and fractionated ethanolic extracts bysilica gel gravity column and HPLC (for details, seeSupplemental Methods and Data File S1). HPLC-QTOF-MS, MS/MS, and NMR data were acquired for partiallypurified metabolites (Supplemental Fig. S1) and com-pared with the Spektraris database.The earliest elutingmetabolite (13.3min)was detected

with a mass-to-charge ratio (m/z) value of 874.2764,which corresponded to a formula of C41H47NO20 for the[M+H]+ ion (Table I). Previous studies that used reverse-phase chromatography with Tripterygium root extractsconsistently listed wilfortrine as the earliest eluting abun-dant peak associated with this formula (Zeng et al., 2013;Cai et al., 2015; Su et al., 2015; Luo et al., 2016). TheMS/MS fragmentation patterns we obtained at a col-lision energy of 30 eV were almost identical to thosereported for wilfortrine when recorded at 25 eV (majorproduct ion at m/z 846; Cai et al., 2015). A search withour 1H-NMR data (isolated metabolite was about 88%pure according to the mass spectrometric estimate)against the Spektraris database returned wilfortrine

(peak 1 in Fig. 4A) as the top hit with an identity scoreof 96% (combined MS and 1H-NMR data; Table I).However, we did not have sufficient material to ac-quire 13C-NMR spectra; therefore, our peak assign-ment is of high confidence but not unambiguous.

A second, less abundant, peak associated with a pre-dicted formula ofC41H47NO20 eluted at 13.9min (peak 2 inFig. 4A). TheMS/MS spectrum of this peak indicated lessfragmentation ([M+H]+ was the peak with highest inten-sity) compared with the data obtained with wilfortrine(Table I).We did not acquireNMRdata for themetaboliteassociated with this peak; therefore, a high-confidenceannotation was not possible.

A third peak corresponding to a predicted formula ofC41H47NO20 was detected at 15.3 min (peak 3 in Fig.4A). Once again, very little fragmentationwas observedat 30 eV (Table I). When the calculated elemental for-mula and 1H-NMR data (generated with a fraction of59% purity according to mass spectrometric estimate)

Figure 2. A, Quantitation of triptolide(white bars) and celastrol (gray bars) inextracts obtained from different Trip-terygium organs. B, Different root types.

Figure 3. Updating the Spektraris resource with NMR spectral recordsof Tripterygium root metabolites.





http://langelabtools.wsu.edu/nmr

http://langelabtools.wsu.edu/nmr




Tab

leI.

Properties

ofmetab

olitesdetectedbyHPLC

-QTOF-MSin

ethan

olicTripterygium

rootex

trac

ts

MS/MSspectrawereac

quired

withafrag

men

tationvo

ltag

eof30eV

.n.a.,Notavailable.

HPLC

Peak

Reten

tion

Time

Form

ula

(HillNotation)

[M+H]+

Error

MS/MSSign

alPatterns

Annotation

Spektraris

Score

Calcu

lated

Observed

1H-N

MR

13C-N

MR

MS/

1H/13C

min

Dppm

%relative

abundan

ce%

iden

tity

112.8

C41H

47NO

20

874.2764

874.2772

0.82

874(2.1),856(18.6),

846(100),828(18.6),

786(4.5),674(12.5),

194(13.4),176(17.0)

Wilfortrine

93

n.a.

96

213.9

C41H

47NO

20

874.2764

874.2770

0.56

874(100),856(6.1),

846(46.0),828(7.2),786(3.6),

674(12.8),194(5.9),

176(18.5)

Unkn

own

n.a.

n.a.

n.a.

315.3

C41H

47NO

20

874.2764

874.2773

1.06

874(100),856(2.3),152(2.5),

134(5.8)

WilfordinineH

65

n.a.

83

Hyp

oglau

nineB

63

n.a.

81

415.8

C38H

47NO

18

806.2866

806.2882

1.94

806(79.8),788(52.4),

746(96.1),704(33.9),

686(33.2),644(22.2),

259(17.0),206(100)

PeritassineA

n.a.

n.a.

n.a.

Wilform

ine

n.a.

n.a.

n.a.

516.3

C41H

47NO

19

858.2815

858.2825

1.17

858(100),840(48.0),

798(22.3),780(11.6),

746(20.0),686(40.4),

206(72.7),178(14.1)

Wilforgine

84

73

86

WilfordinineD

75

73

83

Hyp

oglau

nineD

68

71

80

617.7

C41H

47NO

19

858.2815

858.2828

1.46

858(73.5),840(38.3),

798(63.8),738(26.4),

686(30.8),644(28.9),

206(100),178(29.7)

Hyp

oglau

nineD

64

39

68

Wilforgine

58

37

65

WilfordinineD

57

34

64

718.1

C43H

49NO

18

868.3022

868.3036

1.54

868(100),850(50.0),

808(22.2),746(26.5),

686(44.6),206(60.2),

105(19.1)

Wilforine

n.a.

n.a.

n.a.

819.0

C41H

47NO

19

858.2815

858.2830

1.73

858(72.7),840(100),

798(69.3),756(19.7),

738(24.6),259(27.1),

213(30.9),206(98.9)

WilfordinineD

n.a.

n.a.

n.a.

Hyp

oglau

nineD

n.a.

n.a.

n.a.

Wilforgine

n.a.

n.a.

n.a.

919.7

C29H

36O

6481.2585

481.2587

0.48

245(10.3),231(100),

203(23.4),109(11.1)

Dem

ethylze

ylasteral

100

100

100

10

22.7

C29H

38O

4451.2843

451.2844

0.31

215(5.0),201(100),163(3.5),

158(3.8),123(2.5),109(3.1)

Celastrol

96

90

95


Lange et al.



were searched against the Spektraris database, severalsesquiterpene alkaloid records were returned withhigh identity scores (wilfordinineH [83%], hypoglaunineB [81%], and wilfortrine [81%; assigned to peak 1]).Wilfordinine H and hypoglaunine B are structurallycomplex isomers differing only in the size of the mac-rolide ring (99 methyl group with a contracted ring inhypoglaunine B) and the stereochemistry on the chiralcarbon (C99 in wilfordinine H and C89 in hypoglaunineB; Fig. 4B). Based on the available data, we could notdistinguish these two possibilities.

A peak eluting at 15.8 min (peak 4 in Fig. 4A) waspredicted, based on accurate mass data (m/z value of806.2882), to be consistent with a formula of C38H47NO18(Table I). The fragmentation patterns (abundant pseu-domolecular ion and base peaks atm/z 746 and 206)weresimilar to those reported for two previously character-ized metabolites, wilformine and peritassine A. Theelution order ofTripterygium root constituents in reverse-phase chromatography was in accordance with theproperties reported for peritassine A (Luo et al., 2016;Fig. 4B). However, we did not obtain NMR data for this

Figure 4. Metabolites detected in and purified from ethanolic extracts of Tripterygium roots. A, HPLC-QTOF-MS chromatogram(total ion current) with numbered peaks. B, Structures of identified (or in some cases tentatively identified) metabolites of Trip-terygium roots. The structure of celastrol (peak 10) is shown in Figure 1.





metabolite; therefore, an unambiguous annotation wasnot possible.

A formula of C41H47NO19 was predicted for a peakeluting at 16.3 min (peak 5 in Fig. 4A), a second peak at17.7 min (peak 6 in Fig. 4A), and a third peak at 19 min(peak 8 in Fig. 4A; Table I). The MS/MS fragmentationpattern of peak 5 (pseudomolecular ion as base peak,with an abundant fragment at m/z 206) was closest tothose reported for wilforgine (Luo et al., 2016). Whenthe exact mass (857.274 g mol21) and 1H-NMR spectrafor peaks 5 and 6 were searched against Spektraris (nodata acquired for peak 8), three records were returnedwith almost equally high scores: wilforgine, hypo-glaunine D, and wilfordinine D (Table I). Because of thecomplexity and high similarity of the structures of thesemetabolites, an unambiguous annotation of peaks 5, 6,and 8 was not possible with the available data.

A peak eluting at 18.1 min (peak 7 in Fig. 4A) wasassociated with a predicted formula of C43H49NO18(Table I). The only abundant metabolite commonlyreported with this formula is wilforine (Fig. 4B). Thisannotation also was consistent with the elution orderand fragmentation patterns of sesquiterpene alkaloidsreported previously for reverse-phase HPLC-MS andMS/MS (molecular ion as base peak, with abundantfragments at m/z 206 and 856; Zeng et al., 2013; Caiet al., 2015; Su et al., 2015; Luo et al., 2016). No NMRdata were acquired in this study.

The HPLC-MS properties of a peak eluting at19.7 min (peak 9 in Fig. 4A; retention time and exactmass [480.251 g mol21] and MS/MS fragmentationpatterns [base peak at m/z 231 and additional lowerabundance fragments at m/z 245, 203, and 109]) wereconsistent with those reported for demethylzeylas-teral (Table I; Fig. 4B; Zeng et al., 2013). The samemetabolite record was retrieved with a high score insearches of the exact mass (480.251 g mol21) and 1H-NMR spectral data against Spektraris. However, thepublished 1H-NMR data (Gamlath et al., 1987) were

incomplete, and no 13C-NMR data had been reported.Therefore, we acquired these spectra and updated theSpektraris database accordingly.

The properties of the peak eluting at 22.7 min (peak10 in Fig. 4A; absorptionmaximum at 424 nm; 450.610 gmol21; predicted formula C29H38O4; MS/MS with m/z201 as base peak and smaller fragment peaks atm/z 215,163, 158, 123, and 109) and 1H-NMR spectra corre-sponded to those of an authentic standard of celastrol(Table I; Fig. 1).

Cell Type-Specific Localization of Specialized Metabolitesin Tripterygium Roots Using MSI Technology

The cryosectioning of small Tripterygium woodyroots (diameter 2 mm or less) presented challengesbecause the stele is lined by densely packed pericycleand endodermis layers, which are surrounded by largethin-walled cortex cells, and the cortex cells are lined bya compact periderm. Various trials indicated that con-sistent results could be obtained with a thickness of30mm (with partial rupturing around the stele; Fig. 5A).MALDI-MS was performed with authentic standardsolutions and highly enriched metabolite fractionsspotted onto an imaging target plate, which allowed usto optimize the settings for detection in MALDI-MSIwith tissue samples. For all metabolites (triterpenoidsand sesquiterpene alkaloids), the pseudomolecular ion([M+H]+) and the K+ adduct were detectable at highintensity, while signals for Na+ adducts were generallyof lower abundance (Table II). Ions also were passedthrough the drift tube of themass spectrometer, therebyproviding an additional dimension of separation bydrift time (Table II). Finally, MS/MS experiments wereperformed and fragmentation patterns of signals intissue samples were compared with those obtainedwith standards (Supplemental Fig. S2). In MALDI-MSI,the K+ adduct of celastrol was detected exclusively inthe periderm of Tripterygiumwoody roots (Fig. 5B). The

Figure 5. MALDI-MSI of Tripterygiumroots. A, Root cross section prepared forMALDI-MSI. B, [M+K]+ ion map forcelastrol. C, [M+H]+ ion map of deme-thylzeylasteral. D, [M+K]+ ion map ofwilforine. E, [M+K]+ ion map ofC41H47NO19 (wilforgine, hypoglaunineD, or wilfordinineD). F, [M+K]+ ionmapof C41H47NO20 (wilfortrine, wilfordinineH, or hypoglaunine B).


Lange et al.




signal for demethylzeylasteral ([M+H]+) was consid-erably lower and theMALDI-MSI localization was notas clear cut as that of celastrol, but we still observed apreferential localization to the periderm (Fig. 5C). Incontrast, the K+ adduct signals for sesquiterpenepyridine alkaloids, representing the elemental for-mulas C38H47NO18 (wilforine), C41H47NO19 (wilforgine,hypoglaunine D, and wilfordinine D), and C41H47NO20(wilfordinine H and hypoglaunine B), had only back-ground signal in the stele and periderm, while intensesignals were detected throughout the root cortex (Fig.5, D–F).To obtain independent data on metabolite localiza-

tions (albeit at lower resolution), the outer layers ofTripterygium woody roots (mostly periderm) were re-moved surgically, and the outer and inner root layerswere extracted separately with ethanol and then sub-jected to HPLC-QTOF-MS. In accordance withMALDI-MSI data, triterpenoid quinone methides were detectedat high concentrations in the outer layer (but only assmall peaks in the inner layers), whereas sesquiterpenepyridine alkaloids were present at significantly higherconcentrations in the inner layers compared with theouter layers of the roots (Supplemental Fig. S3).Cross sections of orange-colored roots, obtained us-

ing a vibratome, indicated that the intense color (withconsiderable absorption at 390–430 nm) was restrictedto the periderm (cork cells, cork cambium, and possiblyendodermis; Fig. 6, A and B). Interestingly, these spectralfeatures were consistent with those reported for celastrol(lmax at 256 and 424 nm) and demethylzeylasteral (lmaxat 247, 304, and 388 nm; Harada et al., 1962; Tamakiet al., 1996). We did not observe oil bodies or similarsubcellular structures for the storage of these triterpe-noids (as described previously for diterpenoids in Coleusforskohlii; Pateraki et al., 2014). But how can triterpenoidsbe accumulated to several percent of the periderm layerbiomass?One possibility would be an incorporation into

membranes by displacing (partially or entirely) phyto-sterols. The expectation in this case would be compara-tively low concentrations of phytosterols in the outerlayers of the root. To assess this hypothesis, the outerlayers of Tripterygium roots (mostly periderm) werepeeled off surgically and transferred to glass tubes. Theinner root layers were transferred to separate glass tubes.Following saponification, membrane sterols (primarilyb-sitosterol) were quantified by gas chromatography-MS.The outer cell layers contained b-sitosterol at 1 mmol g21

fresh weight, while the inner layers accumulatedb-sitosterol to 0.47 mmol g21 fresh weight (Fig. 6C),which is evidence against a displacement of membranephytosterols by triterpenoids.It also would be plausible that triterpenoids are ac-

cumulated outside the periderm cells in suberized cellwalls (but not cross-linked with these polymeric mate-rials). Nile Red staining was used to visualize lipophilicregions in root cross sections using confocal laser-scanning microscopy (CLSM). A scan of emissionwavelengths revealed strong fluorescence at both the T

able

II.MALD

I-MSI

detectionofquinonemethidetriterpen

oidsan

dsesquiterpen

epyridinealka

loidsin

Tripterygium

roots

n.a.,Notap

plica

ble;n.d.notdetectable.n=8.

Metab

olite

[M+H]+

Dppm

DriftTime

[M+Na]

+Dppm

DriftTime

[M+K]+

Dppm

DriftTime

ms

ms

ms

Celastrol

451.28406

0.0010

1.746

1.34

89.216

0.40

473.26606

0.0011

1.986

1.12

106.856

0.33

489.24036

0.0010

1.736

0.98

106.086

0.38

Dem

ethylzeylastral

481.25826

0.0007

1.266

0.76

94.836

0.20

n.d.

n.a.

n.a.

519.21516

0.0013

1.956

1.92

109.196

0.35

C38H

47NO

18

806.28626

0.0012

1.316

0.59

141.536

0.50

828.26776

0024

2.566

1.46

147.896

0.38

844.24236

0.0018

1.596

1.37

149.756

0.30

C41H

47NO

19

858.28196

0.0015

1.266

1.15

154.906

3.26

n.d.

n.a.

n.a.

896.23736

0.0026

2.306

1.41

161.516

1.22

C41H

47NO

20

874.27596

0.0029

2.456

2.19

159.616

1.04

n.d.

n.a.

n.a.

912.23296

0.0014

1.006

1.33

161.076

0.62






yellow and red wavelength ranges for stained root sec-tions (Supplemental Fig. S4), indicative of the presence oflipidic materials in the semipolar to neutral range (Fowlerand Greenspan, 1985). The maximum fluorescence in-tensity was emitted from peridermal cell layers in theyellow wavelength range (Supplemental Fig. S4). Redfluorescence (630–670 nm)was emitted by cells of the steleand periderm, while yellow fluorescence (560–600 nm)was localized exclusively to the periderm (Fig. 6, D–F;Supplemental Fig. S5). Sudan IV treatment resulted instaining of the same cell layers (Fig. 6, G and H), whichprovided independent evidence that the periderm cells ofTripterygium roots contain highly suberized walls. Theorange color in bright-field microscopy (due to celastroland demethylzeylasteral accumulation) and yellow fluo-rescence in CLSM (due to suberin deposition) affected thesame periderm cells (Fig. 6, A, D, and G).

DISCUSSION

Development of Combined HPLC-MS and NMR SpectralLibraries Facilitates the Identification of Metabolites inComplex Plant Matrices

Specializedmetabolites purified and characterized frommembers of the genus Tripterygium (family Celastraceae)represent a remarkable structural diversity (SupplementalTable S2). For example, according toour literature searches,105 metabolites have been classified as abietane-type and

an additional 22 as kaurane-type diterpenoids, the vastmajority of which were isolated from roots. Dehydro-b-agarofuran sesquiterpenoids are widely distributed inthe Celastraceae (more than 500 structures) and verycommon in Tripterygium (our searches returned 150unique structures). While sesquiterpene polyestershave been isolated primarily from fruit, sesquiterpenepyridine alkaloid macrolides are present in severalorgans, and their concentrations are often fairly highin roots (Gao et al., 2007). Friedelane triterpenoids area third prominent class of specialized metabolites inthe Celastraceae (more than 50 structures; Shan et al.,2013), and our searches indicated that 26 differentfriedelanes have been described in Tripterygium.

Only a few studies have attempted to quantify spe-cialized metabolites in Tripterygium, and the concentra-tions reported therein varywidely between cultivars andgrowing areas (Zeng et al., 2013; Zhuo et al., 2013; Guoet al., 2014; note that reports aboutmetabolite contents inTripterygium tablets were not considered, because theseare nonstandardized extracts of poorly characterizedor even mixed provenance). There is, however, agree-ment that diterpenoids occur ubiquitously at low levels,with the highest concentration in roots (generally below20 mg g21), which is consistent with the data presentedhere for the diterpene epoxide triptolide. Triterpenoidstend to be much more abundant, and the concentrationof celastrol, the signature metabolite of this class, canexceed 1 mg g21 in roots of mature Tripterygium plants(Zhuo et al., 2013). We detected similar levels in

Figure 6. Accumulation of triterpenoids in Tripterygium root periderm cells with suberized cell walls. A, Cross section of aTripterygium woody root with characteristic orange coloration of periderm. B, Higher magnification of the periderm layer. C,Quantitation of membrane sterols in the outer and inner layers of the root. D, Yellow fluorescence (560–600 nm) from Nile Redlocalizes to the cell walls of periderm cells. E, Bright-field image of the Tripterygium root employed for CLSM in D (control). F,Overlay of images D and E. G, Sudan IV staining of a clarified root cross section. H, A clarified unstained root cross section(control).


Lange et al.








commercial root samples but even higher concentrations(up to 17 mg g21) in roots of young plants (less than4 years since germination). Sesquiterpene alkaloids alsoare prominent specialized metabolites that can reachconcentrations in themilligramper gramof rootmaterialrange (Zeng et al., 2013; Guo et al., 2014).Although triterpenoids and sesquiterpene alkaloids

are quite abundant in Tripterygium, the structural di-versity and lack of access to authentic standards havebeen impediments for more research with this phar-maceutically important genus. Spectral databases canbe invaluable tools to aid in metabolite identification,but their coverage of plant chemical diversity is cur-rently very limited (Johnson and Lange, 2015; Lange,2016). We have been working toward developing da-tabase resources that allow orthogonal searches withHPLC-MS, MS/MS, and NMR data sets. For example,our Spektraris database enabled the rapid identificationof the major taxanes (diverse class of diterpenoids) inTaxus 3 media var Hicksii cell cultures (Cuthbertsonet al., 2013; Fischedick et al., 2015). We have now ex-panded these efforts to include other highly diverseclasses of specialized metabolites (those occurring inTripterygium spp.) that had not been incorporated intoany existing spectral databases. The advantage of ourapproach is that the discovery process is significantlyaccelerated. AMT tag information and MS/MS spectracan be acquired with crude preparations and, for thisproject, provided tentative identifications of severalmetabolite peaks obtained with Tripterygium root ex-tracts. Our database records now also include infor-mation about the occurrence of metabolites acrossspecies, which can be used to further solidify tentativestructural assignments.However, unraveling the identities of isobars (me-

tabolites with the same exact mass), which are commonin Tripterygium, requires additional spectral informa-tion that could only be provided by NMR. One wouldgenerally attempt to further process extracts to fairlyhigh purity to allow unambiguous interpretations ofNMR spectra. We demonstrate that the availability ofan NMR spectral database for all major metaboliteclasses of Tripterygium allows high-confidence struc-tural assignments even when only partially purifiedfractions are obtained in a simple work flow (silica gelgravity column chromatography followed by one-stepHPLC fractionation; Supplemental Methods and DataFile S1). While this approach does not always allow anunambiguous identification of ametabolite (due to verysmall structural variations among isobars that would bedifferentiable only with extensive NMR investigationsoutside the scope of this study), it is very powerful insupporting dereplication efforts (identifying knownmetabolites and excluding these from further consid-eration in phytochemical screening efforts). Adding tothe utility of Spektraris, our search algorithm does notrequire in-depth expertise in NMR spectrum interpre-tation. Users can upload their own spectral recordsthrough a simple Web interface (http://langelabtools.wsu.edu/nmr/submit), thereby contributing to our

efforts to continuously broaden the scope of Spektraris.Finally, in addition to providing access to our spectraldata via our Spektraris online resource, we share ourrecords with both MassBank (Horai et al., 2010) andNMRShiftDB (Steinbeck and Kuhn, 2004) and, as a re-sult, contribute to the expansion of the phytochemicalrepertoire of complementary open-source databases.

MS Imaging Provides Evidence for Cell Type-SpecificDifferences in the Localization of Specialized Metabolitesin Tripterygium Roots

Many of the metabolites that have been isolated fromTripterygium, in particular those accumulating in roots,have been demonstrated to exert considerable toxicityin cell-based assays and animal models (Brinker et al.,2007). Therefore, one would expect that a spatial se-questration is required to prevent similar effects inTripterygium cells. MSI-based approaches allow theevaluation ofmetabolite localization, but plant tissues canpresent notable experimental challenges because of theiranatomical and chemical complexity. The finding that,based on MSI data, celastrol and demethylzeylasteral(the major quinone methide triterpenoids) accumulateprimarily in the peridermwas not a surprise, as these celllayers have a bright orange color in young Tripterygiumroots, which is consistent with the absorption charac-teristics of the above-mentioned triterpenoids. Thehigh specificity of this localization, however, with onlybackground noise being detected for the signature ionsin other cell types, was not necessarily expected. Giventheir location in the outermost cell layers of the root,one may speculate that triterpenoids play importantroles in the first line of defense against bacterial and/or fungal soil pathogens attempting to penetrate roots.Thus far, celastrol has been tested, and found highlyactive, only against gram-positive bacteria with clini-cal pertinence (Moujir et al., 1990), but the relevancefor plant-bacteria interactions has remained unexplored.Fairly broad inhibitory effects against several phytopath-ogenic fungi were reported for both celastrol and its nat-urally occurring methyl ester (Luo et al., 2016). However,these are preliminary studies, and investigations assessingthe ecological significance of Tripterygium triterpenoids,by taking advantage of the natural variability in their ac-cumulation levels in comparative ecological studies,would certainly be desirable.

The concentration of triptolide was too low for con-sistent detection above background by MSI. However,triptophenolide, a diterpenoid that accumulates tosignificantly higher levels in Tripterygium woody roots(150 mg g21), was detectable by MSI. There was a slightenrichment of ions related to this metabolite in theperiderm; however, the background was still fairlyhigh, so this localization needs to be regarded as ten-tative (Supplemental Fig. S6). The measured concen-tration of triptophenolide (0.015% of dry biomassaveraged for the entire root) can thus be regarded as anestimate of the limit of detection by MSI. Compared






http://langelabtools.wsu.edu/nmr/submit

http://langelabtools.wsu.edu/nmr/submit



with triterpenoids, sesquiterpene alkaloids appearedto be more evenly distributed; therefore, the localconcentrations were lower (but ion intensities weresignificantly higher than those of diterpenoids). The high-confidence localization of sesquiterpene alkaloids byMSI, which required a relatively large number of repli-cate experiments (n = 8), indicated a primary occurrencein the root cortex (with only very low levels in the stele) inthis study. MSI was recently employed to unravel thecomplex localization of intermediates and end productsof terpenoid indole alkaloid biosynthesis in several celltypes (phloem-associated parenchyma, epidermis, id-ioblast, and laticifers) of Catharanthus roseus stems(Yamamoto et al., 2016). Magnoflorine, an aporphinealkaloid occurring in Podophyllum spp., was shown,using MSI, to accumulate mainly in the pith and epi-dermis of the rhizome and throughout emerging roots(Marques et al., 2014). These reports are in generalagreement with our finding that alkaloids occur inmultiple cell types in roots. However, MSI studies arejust beginning to shed light on alkaloid localization,and it is too early to generalize such conclusions. Itwould now be instructive to evaluate the localizationof transcripts related to sesquiterpene alkaloid bio-synthesis in Tripterygium, but the genes involved inthis process have not been identified yet. Furthermore,it would also be enlightening to determine the eco-logical roles of sesquiterpene alkaloids, which haveintriguing macrolide structures. The data presentedhere provide the metabolite localization context forthese follow-up investigations.

How Are Triterpenoids Sequestered withinPeriderm Cells?

The high concentration of celastrol in Tripterygiumroots begs the question of how this highly bioactivemetabolite (Salminen et al., 2010) might be segregatedfrom cellular metabolism to avoid cytotoxic effects. Thelocalization of celastrol and demethylzeylasteral tothe periderm of Tripterygium roots is reminiscent of theoccurrence of the diterpenoid forskolin in organellar oilbodies of C. forskohlii (Lamiaceae; Pateraki et al., 2014),which are visible in cross sections by light microscopyand appear to occur only in cork cells. We did not findmicroscopic evidence for the existence of oil bodies incork cells of Tripterygium roots.

A second plausible hypothesis is that triterpenoids inTripterygium roots are incorporated into membranes.Assuming that lipid bilayers constitute about 10% ofthe cell mass (Alberts, 2015), the measured concentra-tion of 18 mg g21 celastrol in orange Tripterygium rootswould translate into a concentration of 180 mg g21 inmembranes. Considering the fact that triterpenoidsaccumulate only in the periderm (which constitutesabout 20% of the volume of orange roots), the con-centration in this layer would be 900 mg g21 (corre-sponding to 90 mass %), which is considerably morethan the highest concentrations estimated for the plant

plasma membrane (50 mass %; Dufourc, 2008). There-fore, one would have to postulate that celastrol replacesother sterols in the plasma membrane of Tripterygiumperiderm cells. However, our measurements indicatedthat the concentration of b-sitosterol, the signature sterolof Tripterygium roots, was in fact higher in the outerlayers compared with the inner layers, which we inter-pret as evidence against the membrane incorporationhypothesis. An explanation for higher rather than lowerconcentrations of b-sitosterol in the outer root layersmight be that the inner layers of the root contain manymetabolically inactive cell types.

Where else could triterpenoids be stored? Bothcelastrol and demethylzeylastral are readily extractedin lipophilic organic solvents, indicating that they arenot glycosylated or in some other way modified (suchas triterpenoid saponins) to facilitate storage in the vacuole(Thimmappa et al., 2014). Triterpenoids in Tripterygiumroots are soluble, as indicated by the rapid loss of orangecolor when roots are immersed in organic solvents. Anextracellular storage also is conceivable, but cellulose(polysaccharide polymer) and lignin (phenolic polymer)might be too polar to serve as the matrix for triterpenoiddeposition. Suberin is a more lipophilic macromolecule ofsecondary cell walls; therefore, we assessed its distributionin Tripterygium roots. Based on microscopic investigationsusing both histochemical stains and CLSM, suberized cellwallswere foundprimarily in theperiderm,whichalsowasthe location of triterpenoid accumulation. Providing directevidence for a colocalization of soluble lipophilic metabo-lites and a lipophilic cell wall polymer is exceedingly dif-ficult, as protocols for visualizing suberin result in theremoval of soluble constituents. It would take significantefforts beyond the scope of this study to develop protocolsaimed at testing the potential absorption of triterpenoids ina suberized cell wall matrix, but it remains the most plau-sible hypothesis consistent with our experimental data.

MATERIALS AND METHODS

Chemicals and Solvents

Triptolide and celastrol were purchased from Cayman Chemical, and stocksolutions were prepared in ethanol (stored at220°C until use). Acetone was ofHPLC grade (Fisher Scientific), ethyl acetate and ethanol were obtained asOmniSolv high-purity reagents (EMD Chemicals), and acetonitrile, methanoland, water were liquid chromatography-MS grade (Sigma-Aldrich). CDCl3 wasobtained from Cambridge Isotope Laboratories. Red phosphorus, a-cyano-4-hydroxycinnamic acid, and 9-anthracenecarboxylic acid were purchased fromSigma-Aldrich. 2,5-Dihydroxybenzoic acidwas sourced from TCIAmerica, andLeu encephalin was from Waters.

Plant Materials

Tripterygium regelii plants were obtained from Woodlanders and maintainedunder greenhouse conditions (illumination, 16-h day, 8-h night (250–500 mE);temperature, 24°C day, 20°C night; relative humidity, 45%–55%). A voucherspecimen was deposited, after careful morphological and phytochemical evalu-ation, with the herbarium of the Field Museum of Natural History in Chicago(collector, D.D. Soejarto). Whole roots of Tripterygium wilfordii were purchasedonline from Chemleader Biomedical. The identity of Tripterygium metaboliteswas established by isolation and purification from whole roots, with subsequenthigh-resolution MS and NMR analyses (Supplemental Fig. S1).


Lange et al.




Tissue Extraction

Root, leaf, or stem material (50 6 0.5 mg) was harvested into liquid nitrogen.Frozen tissue samples were homogenized under liquid nitrogen in a Mixer Mill(MM01; Retsch) using a stainless steel ball in a Teflon receptaclewith a shake rate of20 s21 for 45 s. Finely powdered root homogenate was transferred to 10-mL glasstubes, and metabolites were extracted with 5 mL of acetone for 30 min in an ul-trasound bath (FS30H; Fisher Scientific). Tubes were then centrifuged at 3,000g for5 min, and the supernatant was transferred to another 10-mL glass tube. The sol-vent was removed in vacuo, and the remainder was dissolved in 5 mL of acetonefor a second extraction following the same protocol as listed above. Tissue wasextracted a total of four times, and the finally obtained residue (after solvent re-moval) was dissolved in 1 mL of acetonitrile:water (80:20, v/v) containing 10 mgmL21 internal standard (anthracene 9-carboxylic acid). Extracts were passedthrough a 20-mm filter (polytetrafluoroethylene) and stored at220°C until furtheranalysis. All samples were analyzed within 5 d of preparation.

HPLC-QTOF-MS, MS/MS, and NMR Data Acquisition

The HPLC system (Agilent Technologies) consisted of a G1379B degasser,G1312B binary pump, G1330B thermostat, G1367C autosampler, G1316B col-umn heater, G1315C DAD, and a G1310A isocratic pump (for infusion of massreferences). High-resolution mass spectrometric analyses were performed on a6520 QTOF device (Agilent Technologies) with an electrospray ionizationsource. Separation of Tripterygium metabolites was achieved on a ZorbaxEclipse XDB-C18 Rapid Resolution HT 4.6-mm 3 50-mm 3 1.8-mm columnconnected to a Zorbax SB-C8 Rapid Resolution guard cartridge (2.1 mm 330 mm 3 3.5 mm; Agilent Technologies). The initial conditions were 70% sol-vent A (water with 0.1% [v/v] formic acid) and 30% solvent B (acetonitrile with0.1% [v/v] formic acid). A linear gradient (flow rate of 0.6 mL min21) was usedto increase solvent B to 80% over 35 min, followed by a more rapid gradient to95% solvent B at 40 min. The DAD was set to record at 219, 254, and 424 nm,with spectra being recorded from 200 to 500 nm. To enable a continuous masscorrection, a reference mass solution containing 300 nM purine (exact mass,120.043596 g mol21) and 250 nM hexakis-(1H,1H,3H-tetrafluoropropoxy)-phosphazine (exact mass, 921.002522 g mol21) in acetonitrile:water (95:5, v/v)was infused into the ion source at a flow rate of 0.2 mL min21. The electrospraysource was operated in positive polarity mode at a gas temperature of 325°C, agas flow of 10 L min21, and a nebulizer pressure 2.41 bar. The QTOF m/z scanrange was set to 50 to 1,200 amu, the capillary entrance was maintained at 3,500V, and the MS/MS fragmentor was set to 175 V. MS/MS data were acquired byemploying a collision energy of 30 eV, and electronic files were submitted to theMassBank database (Horai et al., 2010). Data analysis was performed using theMassHunter Workstation software version B.03.01 (Agilent Technologies). Asecond HPLC-QTOF-MS method was used to acquire data to be included in theSpektraris-AMT database as described previously (Cuthbertson et al., 2013).NMR spectra were acquired in CDCl3. A detailed listing of acquisition parame-ters is given in Supplemental Table S3. Images of spectra are provided inSupplemental Figure S1.

Analyte Extraction and HPLC-QTOF-MSMethod Validation

The recovery (R) of target analytes from Tripterygium roots (50 6 0.5 mghomogenate) was determined by (set 1) spiking with 1 mg of triptolide and50 mg of celastrol before extraction, (set 2) spiking with 1 mg of triptolide and50 mg of celastrol after extraction, and (set 3) processing the tissue withoutspiking, which was then taken into account to determine absolute quantities ofanalytes using the following equation:

R ¼ 100 � Peak Area ðSample Spiked Before ExtractionÞ2Peak Area ðSampleÞPeak Area ðSample Spiked After ExtractionÞ2Peak Area ðSampleÞ

The recovery of triptolide and celastrol from T. regelii root extracts was almostcomplete (Supplemental Table S1). The same analyses were used to determinethe reproducibility of the extraction protocol (by comparing replicate data sets)and evaluate matrix effects (by directly comparing detector responses obtainedwith extracts from sets 2 and 3). The reproducibility of the extraction, deter-mined as relative SD (RSD) of three replicate experiments, was 3.7% and 4.7% fortriptolide and celastrol, respectively (Supplemental Table S1). The linear rangeof detection was determined for triptolide and celastrol. The linearity of de-tection was tested for triptolide by injecting 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 50 ng

on column (r2 = 0.99, RSD # 10%) and for the more abundant celastrol byinjecting 1, 50, 100, 500, and 2,500 ng on column (r2 = 0.99, RSD# 6%). The limitof detection (signal:noise ratio of 3:1) was determined as 0.01 and 1 ng on col-umn for triptolide and celastrol, respectively (Supplemental Table S1). The LOQ(signal:noise ratio of 10:1) was recorded as 0.1 and 10 ng on column for trip-tolide and celastrol, respectively (Supplemental Table S1). The LOQ for celastroldetermined with DAD was significantly higher than that for MS (0.2 ng), but,because of the high abundance of the metabolite in T. regelii root extracts,sensitivity was not a concern. The intraday variation of the quantitation was0.9% and 0.6% for triptolide and celastrol, respectively (Supplemental Table S1).The RSD for interday precision, determined by analyte quantitations on threedifferent days, was 6.5% and 0.5% for triptolide and celastrol, respectively(Supplemental Table S1). When a separation using HPLC is combined with MSdetection, matrix effects can lead to ion suppression or enhancement, thusleading to an incorrect quantitation of target analytes. To evaluate the relevanceof such matrix effects for the detection of triptolide (celastrol was quantified byDAD, where matrix effects are insignificant), peak areas were compared forunspiked and spiked extracts from T. regelii roots. Triptolide was present at 0.8mg per 50 mg of root material, while a root extract spiked with 1 mg of triptolidecontained 1.6 mg per 50 mg, indicating that matrix effects were negligible.

Sample Preparation for MSI

Stock solutionsof analytes (0.5mgmL21 in ethanol)weremixed50:50 (v/v)withmatrix 1 solution (1 mg mL21 2,5-dihydroxybenzoic acid in methanol:water[50:50, v/v]) and spotted into standard wells of an imaging target plate (55 341 mm; Waters). A stock solution of Leu enkephalin (1.6 mg mL21 in water) wasmixed 50:50 (v/v) with matrix 2 solution (1 mgmL21 a-cyano-4-hydroxycinnamicacid in acetonitrile:water [70:30, v/v]) and also deposited on the imaging targetplate to be used for lock mass correction (for details, see below).

T. regelii plants were removed from pots, and roots were washed gently withtap water to remove soil. Roots were cut with scissors, placed in 15-mL screw-cap plastic tubes, immediately submerged in liquid nitrogen, and subsequentlyembedded in 3% agarose. These samples were stored at 280°C for up to3 weeks. Directly before analysis, the chamber of a CM 1950 cryostat (LeicaBiosystems) was set to 220°C, and embedded root samples were sectioned to30 mm thickness. Sections were transferred to an imaging target plate, andmatrix 3 solution (40 mg mL21 2,5-dihydroxybenzoic acid in methanol:water[50:50, v/v]) was applied at a flow rate of 0.05 mL min21 with an HTX ImagingSprayer (HTX Technologies) connected to an 1100 Series HPLC Binary Pump(Agilent Technologies). The settings for the sprayer were as follows: nozzletemperature at 80°C; spraying velocity at 1,250 mm min21; 20 passes; dryingtime between passes, 0.3 min; and track spacing of 1 mm.

MSI

A MALDI Synapt G2 mass spectrometer (Waters) was operated in resolutionmode with positive polarity and with a mass range setting ofm/z 100 to 1,000 amu.Data acquisition was performed with MassLynx software version 4.1 (Waters). Asaturated suspension of red phosphorus in acetone was applied to the standardwells of an imaging target plate and used for the mass calibration according to themanufacturer’s instructions. The imaging target plate was introduced into theMALDI sample chamber, and the laser was operated with the following settings:1,000-Hz firing rate; laser energy of 350 (arbitrary units); and a step size of 50 mm.Lock mass correction with Leu enkephalin was repeated every 400 s for 5 s. Ionmobility separationswere performedwith the following settings: heliumgasflowof90mLmin21; trap wave velocity of 311m s21; trap wave height of 4 V; ionmobilitywave velocity of 650m s21; ionmobilitywave height of 40 V; transferwave velocityof 191 m s21; transfer wave height of 0.1 V; and ion mobility wave delay of 450 ms.MS/MS experiments were performed by selection of a precursor mass and a colli-sion energy of 30 eV in the transfer cell. MSI data were processed using the HighDefinition Imaging software version 1.2 (Waters) with lock mass correction.

Microscopy

Roots (approximately 1 mm in diameter) were harvested from greenhouse-grownplants andembedded in 7% (w/v) low-melting-point agarose. Sectioning(30 mm thickness) was performed using a VT 1000 S vibratome (Leica Biosys-tems). Fresh sections were immediately visualized using a BH2 microscope(Olympus). Sections for staining were transferred from water to 100% ethanolusing a gradient and incubated for 1 h to clarify the tissue.













Nile Red Staining

Sectionswere stained in 70%(v/v) ethanol containing0.01%(w/v)NileRed(9-diethylamino-5H-benzo[a]phenoxazine-5-one; Sigma-Aldrich) for 1 h, washedwith 50% (v/v) ethanol, and transferred to water for CLSM.

Sudan IV Staining

Sectionswere stained in 70% (v/v) ethanol containing 0.07% (w/v) Sudan IV[1-(2-methyl-4-(2-methylphenyldiazenyl) phenyl)azonapthalen-2-ol; Sigma-Aldrich] for 3 h, washed with 50% (v/v) ethanol, and transferred to water forbright-field microscopic observation.

CLSM

Imaging was performed using an SP5 confocal laser-scanning microscope(LeicaBiosystems).Tissue sectionswere illuminatedwithanargon laser (488nm,laser intensity setting of 15%). The XYl scan function was employed to deter-mine emission signatures and maxima across the 510- to 775-nm waveband in10-nm steps. Yellow and red fluorescence emission fromNile Red-stained tissuewas detected at 560 to 600 nm and 630 to 670 nm, respectively. Clarified rootsections without staining were used as negative controls.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Spectral data for metabolites isolated from Trip-terygium roots.

Supplemental Figure S2. MALDI-MS/MS spectra for metabolites in Trip-terygium roots.

Supplemental Figure S3. HPLC-QTOF-MS chromatograms of extracts ofthe outer layers and inner layers of Tripterygium roots.

Supplemental Figure S4. Fluorescence emission from a Tripterygium rootcross section scanned across the wave band 510 to 775 nm.

Supplemental Figure S5. Emission of yellow fluorescence and red fluores-cence from Tripterygium roots, with bright-field image and overlay ofbright-field and fluorescence images for comparison.

Supplemental Figure S6. MS-based imaging of triptophenolide in Triptery-gium roots.

Supplemental Table S1. Validation of HPLC-based methods for the quan-titation of triptolide and celastrol.

Supplemental Table S2. Metabolites isolated and characterized frommembers of the genus Tripterygium (literature search).

Supplemental Table S3. NMR acquisition parameters.

Supplemental Methods and Data File S1. Isolation and characterization ofmetabolites from Tripterygium roots.

ACKNOWLEDGMENTS

We thank Washington State University’s NMR Core Facility for access toinstruments and expert support from Dr. Greg Helms as well as Sean Johnsonand Richard Schumaker for incorporating NMR spectral records for Triptery-gium metabolites into the Spektraris database.

Received October 14, 2016; accepted November 13, 2016; published November18, 2016.

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Integrative Approaches for the Identification and Localization of … · Integrative Approaches for the Identiﬁcation and Localization of Specialized Metabolites in Tripterygium