doi.org/10.26434/chemrxiv.8217479.v1

Functionalized Titanium Oxide Nanowire Substrate for Surface-AssistedLaser Desorption/ionization Imaging Mass SpectrometryEwelina P. Dutkiewicz, Han-Jung Lee, Cheng-Chih Hsu, Yu-Liang Yang

Submitted date: 03/06/2019 • Posted date: 04/06/2019Licence: CC BY-NC-ND 4.0Citation information: Dutkiewicz, Ewelina P.; Lee, Han-Jung; Hsu, Cheng-Chih; Yang, Yu-Liang (2019):Functionalized Titanium Oxide Nanowire Substrate for Surface-Assisted Laser Desorption/ionization ImagingMass Spectrometry. ChemRxiv. Preprint.

Imaging mass spectrometry (IMS) is a powerful technique that enables analysis of various molecular speciesat a high spatial resolution with low detection limits. In contrast to the standard matrix-assisted laserdesorption/ionization mass spectrometry (MALDI-MS) approach, surface-assisted laser desorption/ionization(SALDI) is more effective in the detection of small molecules due to the absence of interfering backgroundsignals in low m/z ranges. We developed a functionalized TiO2 nanowire as a solid substrate for IMS oflow-molecular-weight species in biological specimens. We prepared TiO2 nanowires using the inexpensivemodified hydrothermal process and subsequently functionalized it chemically with various silane analogs toovercome the problem of superhydrophilicity of the substrate. Chemical modification changed the selectivity ofimprinting of samples deposited on the surface of the plate and thus improved the detection limits. Due to theenhanced performance, the functionalized TiO2 nanowire substrate could be successfully used for imaging ofcomplex native samples. We applied our new substrate to image distribution of the secondary metabolites in(1) petal of the medicinal plant Catharanthus roseus and (2) microbial co-culture of Burkholderia cenocepacia869T2 vs Phellinus noxius. We observed that secondary metabolites are distributed heterogeneously in apetal, which is consistent with previous results reported for the C. roseus plant leaf and stem. We verified thesemi-quantitative capabilities of the imprinting/imaging approach by comparing results using standard LC-MSanalysis of the plant extracts. Several bacteria-related metabolites produced by B. cenocepacia 869T2 inpresence of P. noxius, which were unable to be detected by MALDI-MS approach, were revealed by our newlydeveloped approach. This suggested that the functionalized TiO2 nanowire substrates-based SALDI is apowerful technique complementary to MALDI-MS.

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Functionalized titanium oxide nanowire substrate for surface-assisted laser

desorption/ionization imaging mass spectrometry

Ewelina P. Dutkiewicz1,2, Han-Jung Lee1, Cheng-Chih Hsu2*, Yu-Liang Yang1*

1Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan

2 Department of Chemistry, National Taiwan University, Taipei, Taiwan

* Corresponding authors

1

ABSTRACT

Detection and localization of low-molecular-weight species in biological specimens provide

important information about biochemical processes taking place in the organism. Imaging

mass spectrometry (IMS) is a powerful technique that enables analysis of various molecular

species at a high spatial resolution with low detection limits. In contrast to the standard 

matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) approach,

surface-assisted laser desorption/ionization (SALDI) is more effective in the detection of

small molecules due to the absence of interfering background signals in low m/z ranges. We

developed a functionalized TiO2 nanowire as a solid substrate for IMS of

low-molecular-weight species in biological specimens. We prepared TiO2 nanowires using the

inexpensive modified hydrothermal process and subsequently functionalized it chemically

with various silane analogs to overcome the problem of superhydrophilicity of the substrate.

Chemical modification changed the selectivity of imprinting of samples deposited on the

surface of the plate and thus improved the detection limits. Due to the enhanced performance,

the functionalized TiO2 nanowire substrate could be successfully used for imaging of complex

native samples. We applied our new substrate to image distribution of the secondary

metabolites in (1) petal of the medicinal plant Catharanthus roseus and (2) microbial

co-culture of Burkholderia cenocepacia 869T2 vs Phellinus noxius. We observed that

secondary metabolites are distributed heterogeneously in a petal, which is consistent with

previous results reported for the C. roseus plant leaf and stem. We verified the

semi-quantitative capabilities of the imprinting/imaging approach by comparing results using

standard LC-MS analysis of the plant extracts. Several bacteria-related metabolites produced

by B. cenocepacia 869T2 in presence of P. noxius, which were unable to be detected by

MALDI-MS approach, were revealed by our newly developed approach. This suggested that

the functionalized TiO2 nanowire substrates-based SALDI is a powerful technique

2

complementary to MALDI-MS.

1. INTRODUCTION

Imaging mass spectrometry (IMS) is a highly versatile and sensitive approach to characterize

and localize molecules in situ in biological specimens. Matrix-assisted laser

desorption/ionization imaging mass spectrometry (MALDI-IMS) is one of the imaging

techniques that enables analysis  across the wide molecular mass range, at high spatial

resolution with low detection limits.1,2 MALDI employs a laser to desorb and ionize analyte

molecules mixed with the excess of the organic matrix, that facilitate the desorption/ionization

process. Typical MALDI matrices are small molecules with high UV light absorption and

good desorption/ionization abilities.3 Those traditional matrices work well for analysis of

larger molecules such as lipids, peptides, and proteins, nevertheless, their use in the analysis

of small molecules is limited due to the presence of interfering matrix-related signals in low

m/z range. To overcome this drawback, various carbon–4, semiconductor–5, and metal–

based6,7 materials have been proposed as alternatives to traditional organic MALDI matrices.8

As a matter of fact, the first approach to use the inorganic matrix for laser

desorption/ionization (LDI) was presented as early as in 1988 by Tanaka et al.9 At that time,

ultra-fine 30-nm size cobalt powder prepared in glycerol as a dispersant was used to analyze

proteins and polymers. Inspired by Tanaka, Sunner at al.4 used much larger 2-150 µm

graphite particles prepared in glycerol for analysis of low molecular weight analytes as well

as peptides and proteins at a much higher resolution. For the first time, the term – graphite

surface-assisted laser desorption/ionization (graphite SALDI) was used. Since then, a large

number of different materials have been reported as SALDI substrates with varying degrees of

performance.10,11

Back in 1988, Tanaka et al.9 have already defined characteristics of the material suitable

to assist LDI, such as strong absorption in the UV range, low heat capacity and large surface

3

area per volume unit. Hence, metal-based materials, comprising chemically stable metal

oxides showed promising performance for SALDI-MS.12 Particularly, TiO2 powder

characterized by its strong UV absorption, mixed with a liquid medium showed the lowest

background noise and the highest intensity during the analysis of low-molecular-weight polar

and non-polar analytes.12 Furthermore, TiO2 nanomaterials were developed to increase the

surface-to-volume ratio and enhance ionization efficiency. Organic dispersive liquid medium

was eliminated to completely reduce interferences in the low mass range below m/z 1000.

TiO2 thin films13,14, nanoparticles15,16, nanotubes17,18, and nanowires19 were developed as very

effective materials for SALDI-MS.

Unlike the other TiO2 nanomaterials, the feasibility of TiO2 nanowires for SALDI-MS

has not been broadly explored due to the inconvenient manufacturing process. Conventional

nanowire synthesis is through the vapor-liquid-solid (VLS) mechanism involving chemical

vapor deposition in the presence of a catalyst under the vacuum.20,21 However, Kim et al. have

recently presented that TiO2 nanowires can be easily synthesized through the modified

hydrothermal process simply by changing aqueous solutions at ambient atmospheric

conditions.19 Nanowires can be synthesized from the planar Ti plate, which is a more

attractive approach for IMS applications. Potentially, TiO2 nanowire surface can be used as a

solid LDI-IMS substrate, although it has a notable superhydrophilic character, in which water

droplets deposited on its surface spreads completely and deteriorate the sensitivity, as well as

the lateral resolution of subsequent IMS.

In this paper, we present TiO2 nanowires prepared on a polished Ti surface through the

modified hydrothermal process as a solid substrate for LDI-IMS for the first time. Importantly,

the TiO2 nanowire surface was chemically functionalized with various silane analogs to

overcome the problem of superhydrophilicity. In addition, chemical modifications changed

the selectivity of imprinting of samples deposited on the surface of the functionalized TiO2

nanowire plate, improving the signal to noise ratios. As a result, our new LDI-IMS substrates

4

showed a greater performance for imprinting and imaging of the distribution of metabolites in

very fragile specimens such as flower petals and agar media. Here, we demonstrate the

imaging of secondary metabolites in medicinal plant Catharanthus roseus petal and microbial

co-culture of Burkholderia cenocepacia 869T2 vs Phellinus noxius.

2. EXPERIMENTAL SECTION

Preparation of functionalized TiO2 nanowire plates

The TiO2 nanowires were synthesized on a polished Ti plate (25 × 75 mm, thickness 0.127

mm, 99.7% trace metal basis, Sigma-Aldrich, Germany) using a modified hydrothermal

process.19 The Ti plates were degreased by soaking in methanol (ACS grade, Macron Fine

Chemicals, USA) for 30 min and dried under a nitrogen stream. The etching process was

carried out by dipping the Ti plates in 10 M potassium hydroxide (BioXtra, ≥85%,

Sigma-Aldrich) prepared in deionized water at room temperature for 24 h with mild shaking.

After alkali solution treatment, the TiO2 nanowire plates were thoroughly rinsed with

deionized water and then dipped in it for 96 h at room temperature with mild shaking. The

deionized water was changed repeatedly every 24 h to efficiently remove alkali residues as

well as etched particles. Finally, the TiO2 nanowire plates were dried under a nitrogen stream

and subjected to heat treatment at 600 °C for 1 h in a muffle furnace (MF-25 P, Enshine

Scientific Corporation, Taiwan).

Following hydrothermal process, the TiO2 nanowire plate was degreased by soaking in

methanol (LC-MS grade, J.T. Baker, USA) for 30 min, dried under a nitrogen stream and

subjected to O2/Ar plasma cleaning process (Solarus model 950, Gatan, USA) for 3 min to

remove any organic contaminants and to generate surface hydroxyl groups for further

chemical modification. The TiO2 nanowire plate was then immediately immersed in silane

analogue solution [10-3 M, TiO2_1: trichloro(3,3,3-trifluoropropyl)silane (97%, Aldrich,

Steinheim, Germany); TiO2_2: trichloro(1H,1H,2H,2H-perfluorooctyl)silane (97%, Aldrich),

5

and TiO2_3: trichlorooctadecylsilane (≥90%, Aldrich)] prepared in n-hexane (Puriss, ACS,

≥99%, Sigma-Aldrich) for 4 h at room temperature. The modified TiO2 nanowire plate was

washed in dichloromethane (Puriss, ACS, ≥99%, Honeywell Riedel-de Haën, Germany) and

consequently in 2-propanol (LC-MS grade, 99.9%, Sigma-Aldrich) for 1 h each time and

dried under a gentle stream of nitrogen. The modified TiO2 nanowire plates were prepared

weekly and stored in the drying cabinet until use.

Preparation of sandwich imprints for petal analysis

C. roseus plants were kept in the plant growth chamber (24 °C, 45% RH, 12 h of light per day).

Fresh flowers were dissected and a single petal was placed on the TiO2 nanowire plate with its

upper surface facing the plate (in one experiment, intact flower was imprinted). Then, the

petal was covered with a silica TLC plate from the top to absorb the excessive liquids leaking

from the petal during imprinting. Bottom of the TiO2 nanowire plate, as well as the TLC plate,

were affixed to the commercial stainless steel plates of the same size with conductive

double-sided tape to ensure electric contact. Petal was manually imprinted using a machine

vise press (MC power vice, VIP 100, Herbert, Taiwan) by applying a load of 1 ton over an

area of 45 × 100 mm for 2 min. Petal tissue residue was immediately removed with tweezers

from the surface of the plate after imprinting. A mixture of calibration standards was

deposited next to the imprint and left for drying in the air for a few minutes before subjecting

the sample for SALDI-IMS analysis. For comparison of different cultivars of C. roseus, a spot

of reserpine (2 µL, 5 mg/L) was deposited next to each imprint as internal standard and

imaged together with the imprint.

Preparation of vinca alkaloid standard solutions and calculation of limits of detection

Standard stock solutions of catharanthine (Cayman Chemical Company, USA), serpentine

(Toronto Research Chemicals, Canada), vindoline (≥98%, Sigma), and vinblastine (≥98%,

6

Sigma) were prepared in amber glass vials in methanol (LC-MS grade, J.T. Baker) and

acetonitrile (LC-MS grade, J.T. Baker, USA) mixture (10:2, v/v) and further diluted with 90%

methanol (LC-MS grade, J.T. Baker). 2 µL of working sample solution was deposited on the

functionalized TiO2_2 nanowire plate. The limit of detection (LOD) was estimated to be equal

to 3-times signal-to-noise ratio (S/N) value. S/N was calculated according to the following

formulae: S/N=2.5×(S-0.5N)/N, where S is the maximum peak intensity, at the center of

specific m/z (for known concentration of a standard compound), N is noise equal to the

amplitude of the signal at the same m/z (±1.0 Da) in blank mass spectrum calculated as

3-times root-mean-square.

SALDI-MS analysis

SALDI-IMS experiments were carried out using autoflex speed MALDI-TOF-TOF mass

spectrometer equipped with the Smartbeam-IITM laser (Bruker Daltonic, Germany) operated

by flexControl (version 3.4) and flexImaging (version 3.0) software (Bruker Daltonic).

Normally, mass spectra were acquired in positive-ion mode, in m/z 160-1500 range with

deflection of the ions below m/z 160. Laser settings were as follows: 1000 Hz repetition rate,

45% intensity (with global attenuation of 20%), “large” laser size corresponding to ~ 100 µm

diameter laser footprint. Mass spectrometer settings were as follows: ion source 1: 19.00 kV,

ion source 2: 16.55 kV, lens: 5.50 kV, reflector: 20.95 kV, reflector 2: 9.50 kV, pulsed ion

extraction: 150 ns. The detector was set to 2190 V, while digitizer was set to 5 GS/s. Each

pixel in the MS image is related to a signal in a single mass spectrum acquired as a sum of

1500 laser shots. The IMS experiments of petal were performed at spatial resolution from 250

to 500 µm. The mass spectrometer was calibrated with the mixture of universal MALDI

matrix (Fluka, St. Gallen, Switzerland), reserpine (Fluka) and peptide calibration standard

(Bruker Daltonic).

7

Additional methods are described in the Supporting Information file.

3. RESULTS AND DISCUSSION

Preparation and characterization of functionalized TiO2 nanowire plate

The TiO2 nanowires were synthesized on a polished Ti plate using the modified hydrothermal

process described before.19,22 As shown in Figure 1a, polished Ti plate was exposed to alkali

treatment at room temperature and then it was thoroughly washed with H2O. The modified

hydrothermal process is a simple procedure carried out under ambient atmospheric conditions

which makes it suitable for mass production. Afterward, TiO2 nanowire plate was subjected to

heat treatment at 600 °C to transform TiO2  lepidocrocite to anatase crystal form.19,23 It has

been already reported that properties of TiO2 nanowires manufactured according to the above

procedure are effective for ionization of various molecules. Pyun’s group prepared arrays of

TiO2 nanowires and applied them to assist analysis of amino acid and peptide standards19,24,

as well as benzylpenicillin in milk25 by LDI-MS. In our study, we adapted the TiO2 nanowires

production procedure and further introduced a chemical modification to prepare the solid

substrate for LDI-IMS (Figure 1a). To verify its efficacy, we applied the new substrate to

imprint and image distribution of the secondary metabolites in a petal of the medicinal plant C.

roseus (Figure 1b) and bacteria/fungi co-culture of B. cenocepacia 869T2 vs P. noxius

(Figure 1c).

We confirmed the successful synthesis of three-dimensional networked structure of TiO2

nanowires through investigating surface morphology by scanning electron microscopy

(Figure 2a, left). Potentially, TiO2 nanowire can be readily used as a solid substrate for IMS,

nevertheless, the wetting properties and surface termination of the substrate have a profound

impact on desorption and ionization efficiency and sensitivity. Due to the superhydrophilic

character of the TiO2 nanowire material, the analyte droplet deposited on this substrate

8

spreads completely on the surface (Figure 2b, left).  A high spreading of the droplet

containing the analyte molecules leads to a significant decrease of sensitivity of detection. To

solve this problem, Pyun’s group prepared an array of multiple TiO2 nanowire zones (300 µm

diameter) on a target plate19. Ti plate was covered with a protective parylene film, then

specific zones were masked with a layer of metal, furthermore plate was subjected to plasma

treatment to expose unmasked Ti, and finally, the metal mask was removed and plates were

etched by KOH solution. After the heat treatment process, parylene coating was removed and

as a result, an array of multiple TiO2 nanowire zones was created.19 Lo et al. stamped TiO2

nanotube layers with small SiO2 loops to restrain sample deposited inside and used them for

analysis of small molecules and peptides.17 SiO2 sol was used as a plaster on the surface of the

nanotube to create a 2-3 mm rings for restricting sample solution within them. Piret et al.

modified TiO2 nanotubes chemically with octadecyltrichlorosilane. At first, the TiO2

nanotube surface was subjected to UV/ozone treatment, then it was reacted with

octadecyltrichlorosilane and treated with UV/ozone irradiation again to adjust

hydrophobicity.18

All the mentioned approaches were developed to overcome the problem of the

superhydrophilic character of TiO2 nanomaterials with the purpose of sample spot deposition,

but not for imaging. Some of them are quite complex procedures and moreover, none of them

were tested specifically for modification of TiO2 nanowires over larger substrate area. In our

work, we simply modified TiO2 nanowire substrate chemically to adapt it for imaging

purposes. Surface of TiO2 nanowire was subjected to O2/Ar plasma to remove any organic

contaminants and to generate surface hydroxyl groups prior to chemical modification with

three different silane analogs: (1) trichloro(3,3,3-trifluoropropyl)silane, (2)

trichloro(1H,1H,2H,2H-perfluorooctyl)silane, and (3) trichlorooctadecylsilane (Figure 1a).

We noticed that the modification with trichlorosilanes was more efficient and robust than with

chlorosilanes, probably due to the higher stability of trichlorosilanes. We confirmed that

9

chemical modification did not affect the three-dimensional networked structure of TiO2

nanowires by scanning electron microscopy (Figure 2a, right). The successful modification

was confirmed by simply depositing a water droplet on the plate and the change of surface

wettability (Figure 2b). Water droplet deposited on the non-modified plate (TiO2_0) spread

completely, while droplets deposited on the modified plates (TiO2_1, TiO2_2, and TiO2_3)

remained with high contact angles. Importantly, we observed that chemical modification

changed the selectivity of imprinting of samples deposited on the surface of the functionalized

TiO2 nanowire plate. As shown in Figure 2c, mass spectral profiles of C. roseus petal

imprinted on four different TiO2 nanowire substrates are drastically different. Although there

is only a minor difference between mass spectra corresponding to imprint on the

non-modified plate TiO2_0 and plate TiO2_1 modified with short trifluoropropyl chain, the

imprinting quality was significantly improved due to its decreased hydrophilicity (Figure S1).

While petal is pressed against a superhydrophilic surface TiO2_0, liquid released from the

plant spreads extensively on the surface that lowers the sensitivity of detection and greatly

affects the resolution of imprinting.

We noticed a high imprinting selectivity towards C. roseus secondary metabolites,

called vinca alkaloids while using plate TiO2_2, functionalized with perfluorooctyl chain

(Figure 2c). We detected high signals at m/z 335.2, 337.2, and 349.2 and lower signal at m/z

457.2 corresponding to the oxidized catharanthine, catharanthine, serpentine, and vindoline,

respectively in a petal imprint (Figure 2c). When using plate TiO2_1, we could also detect

those signals, although their S/N ratios were much lower comparing to plate TiO2_2. Notably,

choosing a nanomaterial with an appropriate affinity for a specific analyte improves detection

limits. When using plate TiO2_3, functionalized with octadecyl chain, we did not detect

signals corresponding to vinca alkaloids, although we could detect many other signals related

to plant metabolites, that could not be observed in blank mass spectra (Figure 2c).

We also verified that the amount of metabolites imprinted on TiO2 nanowire plate and

10

their ionization efficiency is sufficient to perform successful SALDI-MS imaging. We

compared the distribution of petal metabolites imprinted on functionalized nanowire plate

TiO2_3 in assistance of nanomaterial only as well as imprint covered with a standard organic

MALDI matrix (Figure S2). We found that the distribution of vinca alkaloids in the petal

imprint in both cases was very similar, what proves that TiO2 nanowire itself is an effective

substrate for ionization of small molecules. Moreover, when using TiO2 nanowire substrate

only, we could image higher number of small molecules (molecular weight < 800 g/mol),

while in the presence of organic MALDI matrix we observed interfering matrix-related

signals in low m/z (Figure S2). On the other hand, while applying standard MALDI matrix,

signal intensities were higher and we could detect a few molecules of larger molecular

weights, which were not detected while using TiO2 nanowire only.

In another experiment, we tried to enhance ionization efficiency of molecules imprinted

on TiO2 nanowire substrate by supplying H+ ions externally. Trifluoroacetic acid (0.1%) was

sprayed over the petal imprint and we could observe the slight improvement of signal

intensities, however, we could not detect any new signals (Figure S3). Moreover, we also

investigated the possibility to rescan samples in positive and in negative ion modes. TiO2

nanowire plate can possibly act as the universal substrate for analysis in both polarity modes

since this is a matrix-free approach. During the analysis of petal extract deposited on the

surface TiO2 nanowire plate, we detected multiple signals related to plant metabolites ionized

in positive-ion mode. As expected, we could also detect several metabolite signals ionized in a

negative-ion mode such as m/z 165.0, 220.9 or 740.4 (14 signals in total), although a high

background noise derived from the substrate itself was observed (Figure S4).

Application of functionalized TiO2 nanowire substrate for IMS of C. roseus secondary

metabolites in a petal

11

IMS has been profoundly utilized as a valuable tool for molecular spatiotemporal imaging of

natural compounds in plants.26,27 Nevertheless, investigation of the spatial distribution of

low-molecular-weight metabolites is still challenging. As we have mentioned earlier, the

application of a leading imaging technique – MALDI-IMS is limited in this case, due to the

presence of interfering matrix-related signals in low m/z range. Here, we describe a prominent

application of functionalized TiO2 nanowire substrates for IMS of C. roseus secondary

metabolites in a petal. For most of the imaging experiments, plant tissue is cryo-sectioned

(longitudinally or cross-sectioned) into thin slices of 8-50 µm. For MALDI-IMS, plant tissue

section is deposited on a conductive sample carrier (stainless steel or glass covered with

indium tin oxide), dehydrated, covered with organic matrix and dried prior to analysis.27 In

the case of petal analysis, preparation of thin planar longitudinal sections is particularly

difficult since the petal itself is too thin to section. On the other hand, depositing the intact

petal on the sample carrier may affect conductivity and lower ionization efficiency.

Alternatively, flower bud with spirally folded petals can be cross-sectioned and imaged.28

Even so, multiple sections would have to be analyzed and all the results would have to be

merged to reconstruct the distribution of molecules within the whole petal. A relatively simple

approach is to prepare petal imprint and perform the IMS directly on the imprinted substrate.

As such, the surface hydrophobicity and ionization efficient upon laser excitation of the

substrate play crucial roles in obtaining an accurate representation of molecules distribution of

the imprints.

C. roseus, also known as Madagascar periwinkle, is a flowering plant producing a vast

number of secondary metabolites that belong to the group of terpenoid-indole alkaloids, called

vinca alkaloids.29 Two of these alkaloids – vinblastine and vincristine are powerful anticancer

drugs preventing cell division, although they are biosynthesized at very low concentrations.

Vinblastine is produced mostly in plant leaves (but also in flower, stem, seeds, and root) by

coupling two precursors – catharanthine and vindoline. C. roseus is one of the most studied

12

medicinal plants and a model organism for biotechnological studies of vinca alkaloids.29

We imprinted C. roseus petal on functionalized TiO2_2 nanowire substrate and we

detected multiple signals related to vinca alkaloids that were ionized as protonated molecules

[M+H]+ (Figure 3). We detected highly abundant ions at m/z 335.2, 337.2, and 349.2,

corresponding to the oxidized catharanthine, catharanthine, and serpentine, respectively in the

petal imprint. We also detected smaller ion signals at m/z 457.2 and 793.4 corresponding to

vindoline and precursor of vinblastine – anhydrovinblastine. Putative identification of

alkaloids was based on SALDI-MS/MS and HR LC-MS/MS experiments (Figures S5 and

S6). In the case of oxidized catharanthine, catharanthine, serpentine, and vindoline, MS/MS

spectra corresponding to molecules detected in petal extracts were compared with spectra

corresponding to commercial standards. In the case of anhydrovinblastine, due to the lack of

commercial standard, identification was based on a comparison of MS/MS spectra with data

found in the literature.30,31 Besides identified vinca alkaloids, we could detect numerous

sample-related signals in petal imprint.

We investigated the spatial distribution of several vinca alkaloids in dark pink and white

petals of C. roseus flower (Figure 3a-c). In one experiment, we also imaged the whole flower

to compare the distribution of metabolites between petals (Figure 3a). We observed that vinca

alkaloids are not distributed homogeneously in a petal (Figure 3a-c). For example,

catharanthine (m/z 337.2) accumulated in the central and end tips of a petal, while serpentine

(m/z 349.2) was distributed more homogeneously in the center of a petal. In case of vindoline

(m/z 357.2) and anhydrovinblastine (m/z 793.4), tiny amounts were distributed within the

whole petal, while slightly higher amounts were accumulated in the central tip. Distribution of

alkaloids in different petals of the same flower was quite consistent, although the relative

concentration of specific alkaloids was higher in some petals than in others (Figure 3a). Our

results were in agreement with the recent studies, reporting the cell-specific synthesis of vinca

alkaloids in C. roseus stem and leaf leading to low yield during natural biosynthesis of

13

vinblastine.32,33 Here, we reported similar observation for petal revealed by our new method.

Although we could effectively ionize the standard compound of vinblastine (m/z 811.4)

deposited on TiO2 substrate, it was not found in the petal imprint. The possible reason may be

due to the low concentration of this metabolite in a petal or inefficiency in transferring by

imprinting. Moreover, we observed that limit of detection of vinblastine (1.43 ng) deposited

on TiO2_2 is much higher than the limit of detection of other studied metabolites –

catharanthine (0.29 ng), serpentine (0.12 ng), and vindoline (0.21 ng), suggesting that

SALDI-MS using our modified TiO2 substrate are more suitable for compounds of lower

molecular weights.

Interestingly, we observed quite severe oxidation of some vinca alkaloids in LDI-ion

source resulting in the appearance of M-1 signals in positive-ion mode. Initially, we identified

signal at m/z 355.2 as the in-source oxidized form of catharanthine, since we could detect high

signals at m/z 335.2 and 337.2 for the pure standard of catharanthine. The signal at m/z 355.2

was already reported by another group for MALDI-MS experiment of C. roseus plant sections,

but its identity was not revealed.28 Here, we proposed the structure of this species (Figure S7).

Surprisingly, we noticed that the distribution of metabolite at m/z 335.2 in a petal was

different than the distribution of the catharanthine (Figure 3a-c). To verify accuracy of the

imprinting method for semi-quantitative purposes, we prepared extracts (13.3 mg/mL) of

three sections of the petal – (1) end tip, (2) middle part and (3) central tip and subjected them

to SALDI-MS on TiO2 and standard LC-QQQ-MS analysis (Figure 3d, Table S2). Relative

amounts of vinca alkaloids obtained by the two methods were similar (for more results, see

Figure S8). Both datasets were in agreement with imaging experiment and they confirmed

our observations related to the heterogeneous spatial distribution of vinca alkaloids in a petal.

In the cases of the signal detected at m/z 337.2, catharanthine was one of the major

components, however, there was also a contribution of other isomers such as tabersonine and

vindolinine, that was revealed by LC-QQQ-MS analysis (Figure 3d). In the case of

14

anhydrovinblastine, we observed a poor relation of relative amounts of this metabolite in three

petal sections analyzed by SALDI-MS and LC-QQQ-MS. After verification of the accuracy of

the imprinting method for semi-quantitative purposes, we hypothesized that catharanthine was

oxidized at different rates in different regions of the petal before it was harvested for IMS.

This is not surprising, as the other molecular species in the petal may possess anti-oxidative

properties, which could regulate the extent of the oxidation of catharanthine. In addition, the

in-source oxidation may also contribute to the presence of oxidized catharanthine.

Moreover, we investigated the differences in vinca alkaloid profiles between five

cultivars of C. roseus plant (Figure 3e). Four petals of each flower picked from a different

cultivar (Figure S9) were imaged and averaged mass spectra corresponding to each image

were subjected to Partial Least Squares – Discriminant Analysis (PLS-DA). We observed a

clear separation of data points corresponding to five different colors of petals (Figure 3e).

Clusters of data points related to intensively tinted petals – dark pink and dark purple were

closer to each other but far away from the cluster of data points related to white petals.

Clusters of data points related to lightly tinted petals – light purple and pink/white are

between clusters of points related to intensively tinted petals and white petals. Serpentine and

oxidized catharanthine were the most important metabolites responsible for the separation of

clusters in PLS-DA plots. Semi-quantitative comparison of all discussed vinca alkaloids in

different cultivars is presented in Figure 3f. Interestingly, the ratio of oxidized catharanthine

and catharanthine signals in different cultivars was found to be different.

As mentioned above, the distribution of vinca alkaloids in a petal is not homogenous. In

a few particular cases, we observed an interesting large metabolic feature that looked like a

“metabolic loop” in the petal, where we could clearly see the increased accumulation of

several vinca alkaloids in proximity to each other (Figure 4). Since the two crucial precursors

(catharanthine and vindoline) were present at the increased concentrations in close proximity,

we could also observe the increased concentration of the intermediates of the final product in

15

vinblastine biosynthetic pathway (Figure 4a) in this specific region of the petal (Figure 4b).

Remarkably, we were able to detect almost all of the intermediates of the metabolic pathway

leading to the production of vinblastine. The precursors and a final product which give rise to

a “metabolic loop” were thus proposed.

Application of functionalized TiO2 nanowire substrate for IMS of microbial co-culture

Here, we describe a possible application of functionalized TiO2 nanowire substrates for IMS

of microbial co-culture. Burkholderia cenocepacia 869T2 was isolated as an endophyte and is

capable of inhibiting several phytopathogens such as Phellinus noxius, which causes the

brown root rod disease.34 In the IMS analysis of B. cenocepacia 869T2 vs P. noxius co-culture,

interestingly, we found that [M+Na]+ signals at m/z 453.2, 539.4, 625.7, and [M+K]+ signal at

m/z 727.8, corresponding to polymers of poly-(R)-3-hydroxybutyrate (Figure 5 and Figure

S10). Notably, ion signals of poly-(R)-3-hydroxybutyrate polymers were only observed by

SALDI-IMS using the functionalized TiO2_2 nanowire substrate but were not revealed by

MALDI-MS (Figure 5b). The poly-(R)-3-hydroxybutyrate polymers serve primarily as an

energy source and also enhance the resistance of bacterial cells to various stress conditions. In

addition, the unique spatial distribution of signals at m/z 969.2, 1030.6, 1269.2, and 1358.5

implies their production was interfered by the fungal cells in the co-cultural condition.

However, we were not able to detect any signals from fungal cell areas. One of the reasons is

both fungal mycelia and TiO2_2 nanowire substrate are superhydrophobic, therefore the fungal

metabolites were difficult to transfer to the TiO2_2 nanowire substrate for IMS analysis.

CONCLUSIONS

We developed a functionalized TiO2 nanowire as a solid substrate for IMS of

low-molecular-weight species. We prepared TiO2 nanowires through the inexpensive modified

16

hydrothermal process and subsequently functionalized it with various silane analogs. New

TiO2 nanowire substrate was found to be effective to assist LDI-MS as well as IMS of small

molecules. Chemical modification improved detection limits by manipulating the

hydrophilicity of the substrate and the selectivity of imprinting of samples deposited on the

surface of the plate. The modification procedure is straightforward and cost-effective,

allowing flexibility in adjusting the properties of the substrate. Due to the enhanced selectivity,

functionalized TiO2 nanowire substrate could be successfully used for imaging of complex

native biological samples. Moreover, the same substrate can be used for analysis in both

positive and negative ion modes. Furthermore, we successfully applied our TiO2 substrate to

image distribution of the secondary metabolites in (1) petal of the medicinal plant C. roseus

and (2) microbial co-culture of B. cenocepacia 869T2 vs P. noxius. The heterogeneous

distribution of the secondary metabolites in a petal of C. roseus was detected. The

bacteria-related metabolites produced in the presence of fungi were also revealed. We verified

the semi-quantitative capabilities of the imprinting/imaging approach by comparing results

with standard LC-MS analysis of plant extracts. As for now, functionalized TiO2 nanowire is

suitable as a solid substrate for MS of small molecules, where the interferences of the organic

matrix for MALDI-MS are inevitable. We believe that our modified TiO2 substrates will have

great applicability in many other biological and clinical studies.

ASSOCIATED CONTENT

Supporting information

Additional methods (SEM, MALDI-IMS analysis for microbial sample, SALDI-IMS analysis

for microbial sample, SALDI-MS/MS, LC-MS), Table S1 (Optimized settings and retention

times for multiple reaction monitoring (MRM) LC-QQQ-MS method), Figure S1 (Imprinting

of C. roseus petal on functionalized TiO2 nanowire substrates), Figure S2 (Spatial distribution

of metabolites in C. roseus petals – comparison between petal imprint on TiO2 substrate and

17

imprint additionally covered with MALDI matrix), Figure S3 (Spatial distribution of

metabolites in C. roseus petals – comparison between petal imprint on TiO2 substrate and

imprint additionally covered with 0.1% trifluoroacetic acid), Figure S4 (Repeated scans of the

same C. roseus petal extract sample spot in positive- and negative-ion modes), Figure S5

(SALDI-MS/MS spectra of the signal at m/z 335, 337, 349, and 457), Figure S6

(LC-HR-MS/MS spectra of white petal extract), Figure S7 (MS/MS fragmentation pattern of

catharanthine and oxidized catharanthine), Figure S8 (Comparison of relative amounts of

vinca alkaloids in extracts from three sections of white petal analyzed by SALDI-MS and

LC-QQQ-MS), Figure S9 (Flowers of five cultivars of C. roseus plant investigated in the

study), Figure S10 (LC-HR-MS/MS spectra of poly-(R)-3-hydroxybutyrate from the

microbial extract).

AUTHOR INFORMATION

Corresponding Author

*E-mail: Y.-L. Yang: ylyang@gate.sinica.edu.tw

C.-C. Hsu: ccrhsu@ntu.edu.tw

ORCID: Y-L Yang: 0000-0002-3533-5148

C.-C. Hsu: 0000-0002-2892-5326 

Author Contributions

E.P.D. prepared TiO2 nanowire substrate, optimized analysis conditions and collected most of

the data included in the manuscript, E.P.D. and H.-J.L. developed and optimized conditions of

TiO2 nanowire substrate preparation, E.P.D, C.-C.H, and Y.-L.Y designed experiments,

reviewed the data and wrote the manuscript.

Notes

The authors declare no competing financial interest.

18

ACKNOWLEDGMENTS

This research was supported by Ministry of Science and Technology (MOST), R.O.C. (Grant

nos.: 104-2320-B-001-019-MY2, 107-2321-B-001-038- and 108-2636-M-002-008-), and

Center for Emerging Materials and Advanced Devices, National Taiwan University (NTU).

We thank W.-C. Lai, Surface and Nanoscience Laboratory, Institute of Physics, Academia

Sinica for taking SEM images, H.-J. Huang, ASCEM, Academia Sinica, for the assistance and

use of the Solarus plasma cleaner, and Metabolomics Core Facility, Agricultural

Biotechnology Research Center (ABRC), Academia Sinica, for performing LC-QQQ-MS and

LC-HR-MS analysis of petal extracts. We also thank B.-W. Wang, ABRC, Academia Sinica,

for assistance during TiO2 nanowire substrate preparation, H.-D. Cheng, ABRC, Academia

Sinica, for taking care of C. roseus plants and Y.-M. Lai, ABRC, Academia Sinica, for

collecting the IMS data of microbial co-culture samples.

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23

FIGURES

Figure 1. Preparation protocol of functionalized TiO2 nanowire substrate and its applications. a) Major steps during

preparation of the TiO2 nanowire substrate. b) Application of the TiO2 nanowire substrate for IMS of C. roseus petal. c)

Application of the TiO2 nanowire substrate for IMS of the microbial co-culture of Burkholderia cenocepacia 869T2 vs

Phellinus noxius.

24

Figure 2. Characterization of the functionalized TiO2 nanowire substrate. a) Scanning electron microscopy images of

nanowires created on the surface of the polished Ti plate before (left) and after (right) chemical modification (magn. 30,000).

b) Optical images of a water droplet deposited on the surface of TiO2 nanowire substrate before (first left) and after

modification with various silanes (top and side views). c) Average mass spectra related to images of C. roseus petal imprinted

on four different TiO2 nanowire substrates. Signal intensities were normalized with respect to total ion currents.

25

Figure 3. Spatial distribution of vinca alkaloids in C. roseus petals. Distribution of alkaloids in a) intact small flower, b)

single dark pink petal, and c) single white petal of a bigger flower. Signals at m/z 335.2, 337.2, 349.2, 457.2, and 793.4

correspond to the oxidized form of catharanthine, catharanthine, serpentine, vindoline, and anhydrovinblastine, respectively.

Raster step was 250 µm. The color bar represents MS signal intensity. d) Comparison of relative amounts of vinca alkaloids

in extracts from three sections of white petal analyzed by SALDI-MS and LC-QQQ-MS. C – catharanthine, T – tabersonine,

V – vindolinine. Data were scaled for comparison. Error bar: SD (3 technical replicates) e) Profiling of petals from five

different cultivars of C. roseus plant. (PLS-DA scores plot, four petals of each color were imaged). f) Comparison of relative

amounts of vinca alkaloids in petals of five different cultivars of C. roseus plant. Colors: DP – dark pink, DPP – dark purple,

LPP – light purple, PW – pink/white, W – white. Error bar: 1.5 SD (four biological replicates). All the signal intensities were

normalized with respect to total ion currents (except (e), where signal intensities were normalized with respect to IS signal

intensity).

26

Figure 4. The special features of metabolites observed in C. roseus petal. a) Biosynthetic pathway of vinblastine. b) MS

images of C. roseus petal imprint. Signals at m/z 337.2, 369.2 2, 335.2 2, 457.2 2, 791.4 2, 793.4 2, and 811.4 2 correspond to

catharanthine, intermediate of catharanthine, the oxidized form of catharanthine, vindoline, an intermediate of

anhydrovinblastine, anhydrovinblastine, and vinblastine, respectively. Notice, that vinblastine was not observed due to its low

concentration in the petal. Raster step was 250 µm. Signal intensities were normalized with respect to total ion currents. The

color bar represents MS signal intensity.

27

Figure 5. Spatial distribution of bacterial metabolites in the co-culture of Burkholderia cenocepacia 869T2 vs Phellinus

noxius. The white colony in the center is B. cenocepacia 869T2, and the white mycelia on the right are P. noxius. a) Selected

signals related to the bacterial metabolites. Signals at m/z 453.2, 539.4, 625.7, and 727.8, correspond to

poly-(R)-3-hydroxybutyrate. Raster step was 1000 µm. The color bar represents MS signal intensity. b) Average mass spectra

(m/z 180-840) of MALDI-IMS and SALDI-IMS of the co-culture of B. cenocepacia 869T2 vs P. noxius. The signals of

poly-(R)-3-hydroxybutyrate shown in b) are highlighted. Signal intensities were normalized with respect to total ion currents.

28

download fileview on ChemRxivFunctionalized titanium oxide nanowire substrate for surfac... (2.36 MiB)

Supporting information

Functionalized titanium oxide nanowire substrate for surface-assisted laser

desorption/ionization imaging mass spectrometry

Ewelina P. Dutkiewicz1,2, Han-Jung Lee1, Cheng-Chih Hsu2*, Yu-Liang Yang1*

1Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan 2 Department of Chemistry, National Taiwan University, Taipei, Taiwan

SUPPLEMENTARY METHODS

SEM

The morphology of the nanowires was characterized using a scanning electron microscope

instrument (Inspect F, FEI, Japan) operated at 5 kV.

MALDI-IMS analysis for microbial sample

For the microbial co-culture sample, the 10 μL of Burkholderia cenocepacia 869T2 broth was

added to the PDA agar plate and the 8 mm Phellinus noxius mycelia plug from a fresh

growing fungal plate (7 days) was placed at the opposite edge of the spot at a 1.5 cm distance.

After 4 days of incubation, areas of agar media containing the microbial colonies and fungal

mycelia were excised from the Petri dish and transferred onto the MALDI stainless steel

target plate. The universal MALDI matrix (Fluka, St. Gallen, Switzerland) was spread on top

of the agar by using HTX TM-SprayerTM (HTX Technologies LLC, USA). The parameters

used for each agars were 1200 mm/min velocity, 0.1 mL/min flow rate, 12 passes, 3 mm line

spacing and a nozzle temperature of 80 °C. Once the sample was completely covered with

matrix, it was exposed in a 37 °C incubator for overnight until it was deemed dried. MALDI-

IMS was collected using an autoflex speed MALDI-TOF-TOF mass spectrometer equipped

with the Smartbeam-IITM laser (Bruker Daltonic, Germany) operated by flexControl (version

3.4) and flexImaging (version 3.0) software (Bruker Daltonic). The mass spectra were

acquired in positive-ion mode, in m/z 100–2000 range. Laser settings were as follows: 500 Hz

repetition rate, 25% intensity (with global attenuation of 20%), “large” laser size

corresponding to  ~ 100 µm diameter laser footprint. Mass spectrometer settings were as

1

follows: ion source 1: 19.00 kV, ion source 2: 16.6 kV, lens: 8.7 kV, reflector: 21.0 kV,

reflector 2: 9.4 kV, pulsed ion extraction: 150 ns. The detector was set to 2190 V, while

digitizer was set to 5 GS/s. Each pixel in the IMS is related to a signal in a single mass

spectrum acquired as a sum of 1000 laser shots. The IMS experiments were performed at a

spatial resolution of 1000 µm. The mass spectrometer was calibrated with the mixture of

universal MALDI matrix (Fluka, St. Gallen, Switzerland) and peptide calibration standard

(Bruker Daltonic).

SALDI-IMS analysis for microbial sample

The areas of agar media containing the microbial colonies and fungal mycelia were excised

from the Petri dish and transferred onto the TiO2 nanowire plate with its upper surface

(microbial cells) facing the plate. The microbial samples were manually imprinted by

applying a load of one 500 mL beaker for 2 min. The microbial samples were immediately

removed with tweezers from the surface of the plate after imprinting. SALDI-IMS

experiments were carried out using autoflex speed MALDI-TOF-TOF mass spectrometer

equipped with the Smartbeam-IITM laser (Bruker Daltonic, Germany) operated by flexControl

(version 3.4) and flexImaging (version 3.0) software (Bruker Daltonic). The mass spectra

were acquired in positive-ion mode, in m/z 60-2500 range. Laser settings were as follows: 500

Hz repetition rate, 80% intensity (with global attenuation of 20%), “large” laser size

corresponding to  ~ 100 µm diameter laser footprint. Mass spectrometer settings were as

follows: ion source 1: 19.00 kV, ion source 2: 16.55 kV, lens: 5.50 kV, reflector: 20.95 kV,

reflector 2: 9.50 kV, pulsed ion extraction: 150 ns. The detector was set to 2190 V, while

digitizer was set to 5 GS/s. Each pixel in the MS image is related to a signal in a single mass

spectrum acquired as a sum of 1000 laser shots. The IMS experiments were performed at a

spatial resolution of 1000 µm. The mass spectrometer was calibrated with the mixture of

universal MALDI matrix (Fluka, St. Gallen, Switzerland) and peptide calibration standard

(Bruker Daltonic).

SALDI-MS/MS

SALDI-MS/MS experiments were carried out in the LIFT mode using the same MALDI-

TOF-TOF instrument as for SALDI-MS and MALDI-MS experiments. Mass spectra were

acquired in positive-ion mode. Laser settings were as follows: 200 Hz repetition rate, 45%

intensity (with global attenuation of 20%), “large” laser size corresponding to ~ 100 µm

diameter laser footprint. Mass spectrometer settings were as follows: ion source 1: 6.00 kV,

2

ion source 2: 5.30 kV, lens: 3.00 kV, reflector: 27.00 kV, reflector 2: 11.70 kV, Lift 1: 19.00

kV, Lift 2: 4.80 kV, pulsed ion extraction: 120 ns. The detector was set to 2225 V, while

digitizer was set to 5 GS/s. In fragmentation mode, detector gain boost was set to 180%, laser

power boost to 100%, and analog offset to -1.0%. PLMS was “on”. The isolation window was

set to -3+1 Da for parent ion at m/z 335.2, -1+3 Da for parent ion at m/z 337.2, and -3+3 for

remaining parent ions. MS/MS spectra presented in Figure S5 were acquired as a sum of

1500 laser shots in parent-ion mode and 1500 shots in fragment-ion mode.

LC-MS

LC-Orbitrap-MS

The ACQUITY ultra-high performance liquid chromatography system (Waters, USA), fitted

with ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm; Waters), was used to

separate vinca alkaloids. Mobile phase A consisted of 0.1% formic acid in the water, while

mobile phase B consisted of 0.1% FA in acetonitrile. Gradient elution was as follows: 95% A

at 0 min, 0.5% A at 6 min, 0.5% A at 8 min, 95% A at 8.2 min, and 95% A at 10 min. The flow

rate was kept at 0.4 mL/min, column temperature was set to 40 °C, injection volume was 10

µL. The Orbitrap Elite mass spectrometer equipped with HESI ion source (Thermo Fisher

Scientific, USA) was used as a detector. MS was operated in a positive-ion mode in m/z 100-

1500 range at 15000 resolution. Source voltage was set to 3.5 kV.

LC-QQQ-MS

The ACQUITY ultra-high performance liquid chromatography system (Waters), fitted with

ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm; Waters), was used to

separate vinca alkaloids. Mobile phase A consisted of 0.1% formic acid in the water, while

mobile phase B consisted of 0.1% FA in acetonitrile. Gradient elution was as follows: 95% A

at 0 min, 0.5% A at 6 min, 0.5% A at 8 min, 95% A at 8.2 min, and 95% A at 10 min. The flow

rate was kept at 0.4 mL/min, column temperature was set to 40°C, injection volume was 1 µL.

The Xevo TQ-S mass spectrometer (Waters) equipped with ESI ion source was used as a

detector. MS was operated in positive-ion mode. Source voltage was set to 3.3-3.4 kV.

Optimized settings and retention times for multiple reaction monitoring (MRM) are shown in

Table S1.

3

SUPPLEMENTARY TABLES

Table S1. Optimized settings and retention times for multiple reaction monitoring (MRM)

LC-QQQ-MS method.

Compound Collision voltage (V) Monitored transition RT (min.)

Anhydrovinblastine 40 793 > 563 2.88

Ajmalicine 20 353 > 210 2.61

Catharanthine 15 337 > 173 2.69

Oxidized catharanthine 15 335 > 228 2.66

Reserpine (IS) 30 609 > 397 3.32

Serpentine 35 349 > 261 2.66

Tabersonine 30 337 > 180 2.60

Vinblastine 50 811 > 224 2.69

Vindoline 50 457 > 173 2.88

Vindolinine* 20 337 > 320 2.25

19S Vindolinine* 20 337 > 320 2.15

Yohimbine 20 355 > 212 2.29

*Vindolinine and 19S Vindolinine were quantified together (peak areas were summed up).

4

SUPPLEMENTARY FIGURES

Figure S1. Imprinting of C. roseus petals on functionalized TiO2 nanowire substrates. Images were taken before removing petal residues after imprinting. Arrows indicate liquid released from the petal and spread extensively on a highly hydrophilic surface.

5

Figure S2. Spatial distribution of metabolites in C. roseus petals – comparison between petal imprint on TiO2 substrate (TiO2 only) and imprint additionally covered with MALDI matrix (TiO2 and matrix). a) Optical images of white and dark pink petals and their imprints on TiO2_2 plate, b) distribution of vinca alkaloids discussed in the main text, c) distribution of metabolites for which ionization was enhanced in presence of matrix, d) distribution of metabolites for which images can be obtained with TiO2 only, e) distribution of other interesting metabolites related to petal color (the same result for TiO2 only and TiO2 and matrix). Imprints were covered with a layer of universal MALDI matrix (10 mg/mL) by HTX TM-SprayerTM (one pass, 0.1 mL/min, lines 1 mm away; HTX Technologies LLC, USA). Raster step was 400 µm. Signal intensities were normalized with respect to total ion currents.

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Figure S3. Spatial distribution of metabolites in C. roseus petals – comparison between petal imprint on TiO2 substrate (TiO2 only) and imprint additionally covered with 0.1% trifluoroacetic acid (TiO2 and TFA). a) Optical images of white and dark pink petals and their imprints on TiO2_2 plate, b) distribution of vinca alkaloids discussed in the main text, c) distribution of metabolites for which ionization was enhanced in presence of TFA. Imprints were covered with a layer of TFA (0.1% in 90% methanol) by HTX TM-SprayerTM (one pass, 0.1 mL/min, lines 1 mm away; HTX Technologies LLC, USA). Raster step was 400 µm. Signal intensities were normalized with respect to total ion currents.

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Figure S4. Repeated scans of the same C. roseus petal extract sample spot in positive- and negative-ion modes. Raw mass spectra for blank and white petal extract (50 mg/mL) sample spot collected in a) positive- and b) negative-ion modes deposited on TiO2_2 plate. c) Images of sample spots of white and dark pink petal extracts. Signals related to plant metabolites detected in negative-ion mode – some of the signals are specific to the color of petal. All new signals are highlighted in spectra presented in b). Raster step was 400 µm. Signal intensities were normalized with respect to total ion currents.

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Figure S5 a-b. SALDI-MS/MS spectra of the signal at m/z 335. a) Mass spectra scaled to a base peak, b) magnified spectra. Upper spectra correspond to catharanthine standard (10 mg/L), while lower spectra correspond to white petal extract (50 mg/mL) deposited on TiO2_2 plate. Spectra collected in positive-ion mode.

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Figure S5 c-d. SALDI-MS/MS spectra of the signal at m/z 337. a) Mass spectra scaled to a base peak, b) magnified spectra. Upper spectra correspond to catharanthine standard (10 mg/L), while lower spectra correspond to white petal extract (50 mg/mL) deposited on TiO2_2 plate. Spectra collected in positive-ion mode.

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Figure S5 e-f. SALDI-MS/MS spectra of the signal at m/z 349. a) Mass spectra scaled to a base peak, b) magnified spectra. Upper spectra correspond to serpentine standard (10 mg/L), while lower spectra correspond to white petal extract (50 mg/mL) deposited on TiO2_2 plate. Spectra collected in positive-ion mode.

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Figure S5 g-h. SALDI-MS/MS spectra of the signal at m/z 457. a) Mass spectra scaled to a base peak, b) magnified spectra. Upper spectra correspond to vindoline standard (10 mg/L), while lower spectra correspond to white petal extract (50 mg/mL) deposited on TiO2_2 plate. Spectra collected in positive-ion mode.

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Figure S6. HR LC-MS/MS spectra of white petal extract. MS/MS spectra of signals at a) m/z 335.2, b) m/z 337.2, c) m/z 349.2, d) m/z 457.2, and e) m/z 793.4. Spectra collected in positive-ion mode.

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Figure S7. MS/MS fragmentation pattern of catharanthine and oxidized catharanthine.

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Figure S8. Comparison of relative amounts of vinca alkaloids in extracts from three sections of white petal analyzed by SALDI-MS and LC-QQQ-MS. Signals at m/z 353.2, 355.2, and 811.4 correspond to ajmalicine, yohimbine, and vinblastine, respectively. Data were scaled for comparison. Error bar: SD (three technical replicates).

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Figure S9. Flowers of five cultivars of C. roseus plant investigated in the study.

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Figure S10. HR LC-MS/MS spectra of poly-(R)-3-hydroxybutyrate from the microbial extract. MS/MS spectra of signals at a) m/z 453.2, b) m/z 539.2, c) m/z 625.2, d) and m/z 727.3. Spectra collected in positive-ion mode. 

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