2012 Pavarini - Application of MALDI-MS Analysis of Rainforest

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Application of MALDI-MS analysis of Rainforest

chemodiversity: a keystone for biodiversityconservation and sustainable use

Daniel P. Pavarini,a Denise B. da Silva,b Carlos A. Carollo,c

Amanda P. F. Portella,a Sabrina R. Latansio-Aidar,d Pedro O. Cavalin,d

Viviane C. Oliveira,d Bruno H. P. Rosado,d† Marcos P. M. Aidar,e

Vanderlan S. Bolzani,h Norberto P. Lopesa* and Carlos A. Jolyf,g*

Brazil hosts the largest proportion of global biodiversity[1]

and hasdemonstrated its commitment in conservation and sustainable

use being a key negotiator of the Nagoya Protocol. The “Convention

on Biological Diversity” (CBD) calls for actions to reduce extinction

rates, something that according with different theories[2,3] is of

fundamental importance for the survival of life on Earth. Contrary

to its position in the CBD meetings, Brazil approved a new Forest

Code that will result in escalating deforestation,[4] increasing the

urgency to demonstrate the value of native species.

Extractive-based activities of forest inhabitants are basically

economical prospective activities upon the forest richness whose

act harmfully against the environment. For centuries, such

activity resulted in low profits, triggering a perverse logic that

profit increase is necessarily linked to extraction increase.[5] In

the last decades, new strategies, as for instance the Sustainable

Development Reserve Mamiraua,[6] are showing that forest

preservation can also be profitable.

Considering the new paradigm of green economy,[7] which

now surrounds all this tensioned discussion, we are bringing to

the eyesight of policy-makers results on biodiversity conservation

research: combining floristic[8] and chemodiversity surveys, using

fast high throughput mass spectrometry screening (HT-MSS), to

screen forest leaves for economically valued natural products.

Matrix-assisted laser desorption/ionization (MALDI) ionization-

based machines are well known by their ability to furnish fast

data[9] and can be an important tool for HT-MSS.

One of the aims of a long-term ongoing BIOTA/FAPESP[10]

research project at the Serra do Mar State Park is to understandecophysiological traits of leaves, and MALDI-MS was an alterna-

tive to identify alkaloids as one of the nitrogen sink. Regarding

the following main points: (1) analysis of plant attributed nitro-

gen fixation and (2) profitable policies for biodiversity conserva-

tion, we suggest the screening for alkaloids to be carried out

using the leaves of the trees that are top ranked in population

density lists within the forest sociology. In order to reach these

aims, a set larger than 500 samples was screened for the occur-

rence of alkaloids and botanical identification. Classical chemical

procedures were also applied to validate the results (Dragendorff,

Mayer and Wagner assays). In this letter, we provide the first HT

MALDI-MS and MALDI-MS/MS forest screening method to

provide added value to local specimens of plants. Ionic liquid

was used to get around known ionization problems of smallmolecules (< 1200 Da) by avoiding isobaric ions formation by

the matrix. Although, LC-ESI-MS is the common technique to

screen small molecules, it is highly time consuming, since in six

days, only 50 samples can be analyzed.[11] Therefore, our strategy

is based on the ability of MALDI to quickly screen a very large

number of samples using an ionic liquid as matrix. Using this ap-

proach, we can screen up to 200 samples within a 3 h time-frame.

* Correspondence to: Norberto P. Lopes, Núcleo Pesquisas em Produtos Naturais

e Sintéticos, Departamento de Física e Química, Faculdade de Ciências

Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Zipcode 14040-903,

Ribeirão Preto, SP, Brazil. E-mail: [email protected]

* Carlos A. Joly, Plant Biology Department, Biology Institute, State University of Campinas/UNICAMP, POBox6109, ZIPCode 13083-970 Campinas, SP, Brazil. E-mail:

[email protected]

† Present Address: Instituto de Pesquisas Jardim Botânico do Rio de Janeiro,

Diretoria de Pesquisas. 22460-030- Rio de Janeiro, RJ – Brazil

a Núcleo Pesquisas em Produtos Naturais e Sintéticos, Departamento de Física e

Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade

de São Paulo, Zipcode 14040-903, Ribeirão Preto, SP, Brazil

b Lychnofl ora, Pesquisa e Desenvolvimento em Produtos Naturais, Universidade

de São Paulo, Zipcode 14040-903, Ribeirão Preto, SP, Brazil

c Departamento de Farmácia-Bioquímica, Centro de Ciências Biológicas e da

Saúde, Universidade Federal de Mato Grosso do Sul, POBox 549, ZIPCode

79070-900, Campo Grande, MS, Brazil

d Programa de Pós-Graduação em Biologia Vegetal, Universidade Estadual de

Campinas, Zipcode 13083-970, Campinas, SP, Brazil

e Núcleo de Pesquisa em Fisiologia e Bioquímica, Instituto de Botânica/SMA,

POBox 68042, ZIPCode 04045-972, São Paulo, SP, Brazil

f Plant Biology Department, Biology Institute, State University of Campinas/UNI-

CAMP, POBox 6109, ZIPCode 13083-970, Campinas, SP, Brazil

g Secretary of Policies and Programs in Research and Development, Ministry of

Science, Technology and Innovation, Esplanada dos Ministérios, Brasília, Brazil

h Instituto de Química de Araraquara, Departamento de Química de Química

Orgânica, NuBBE, Universidade Estadual Paulista, POBox 355, ZIPCode

14800-900, Araraquara, SP, Brazil

J. Mass Spectrom. 2012, 47 , 1482–1485 Copyright © 2012 John Wiley & Sons, Ltd.

JMS letters

Received: 21 July 2012 Revised: 31 August 2012 Accepted: 4 September 2012 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jms.3100



This is a 192-fold increase in throughput, and therefore it can

be ef ficiently used to prioritize plants for candidate added

value components. Finding plants with the correct signatures,

indicating candidate molecules that have commercial value, is

then prioritized for the lower throughput but quantitative LC-MS

studies where the retention time, UV and tandem MS signatures

Figure 1. Scheme to ensure Forest Inhabitants economical return of sustainable exploitation of chemodiversity.

306.2092

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313.2895

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Figure 2. Mass spectra of the extract of Simira sampaioana obtained by MALDI-TOF/TOF (A: without matrix, B: with DHB matrix, C: with DHB and CTABmatrix, D: with the ionic liquid matrix produced from DHB and triethylamine, E: MS/MS spectrum of strictosidine. All the illustrated spectra were done inpositive mode) and the chromatogram at 270 nm obtained by LC-DAD-MS of the extract of S. sampaioana and the chemical structure of strictosidine (F).

JMS letters

J. Mass Spectrom. 2012, 47 , 1482–1485 Copyright © 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jms



are used to confirm the identity of the molecule (Figs. 1 and 2).

Thus, the screening of an entire forest can be accomplished in a

short time, which is currently not feasible with other

methodologies.

Our current forest dereplication using all plants obtained in

floristic survey[8] (over 1000 entries) showed alkaloids in leaves

only in four species Rollinia sericea, Guatteria gomeziana, Ocotea

sp and Simira sampaioana. These data are in agreement with

the classical natural products concepts,[12] but is important to

define the economical perspectives. Only 5% of the selected

species showed the presence of alkaloids, suggesting a low

N-fixation through secondary metabolite storage. For economi-

cally valued natural products perspective, the HT MALDI

analysis revealed high levels of alkaloids on Simira sampaioana

(Rubiaceae) leaves by the presence of strictosidine as the major

metabolite (Fig. 2). The high-resolution data and the MS/MS data

are in agreement with the previous published data. [13] Strictosi-

dine is the starter of indolic alkaloid synthesis, such as Vincristine.

In 2010, the revenue of the main supplier of Vincristine was of the

order of 16 billion dollars.[14] The occurrence of such high levels

of its key biosynthesizer represents a great opportunity for forest

inhabitants to profit on yielding leaves’

compounds by singlesolvent-recrystallization methods and profit by supplying

“Research Spin-off ” companies with the yielded compounds. For

this, first, some extracts, obtained from plants, were analyzed

by MALDI without matrix (LDI), but it was not possible to identify

the major compounds from many extracts due to the extensive

fragmentation and/or degradation of compounds. Therefore, dif-

ferent matrices were tested, and the best matrix was ionic liquid

(Fig. 2). The matrix plays a main role in the ionization and desorp-

tion processes, such as the absorption of laser energy avoiding

the degradation of analytes and facilitating the charge transfer

into the gas phase by intermolecular interaction reduction.[15]

Such trait outlines matrix usage needs. Regular and commercial

available matrices necessarily yield many ions in the same mass

range of our targeted compounds or even isobaric ions.[16] Upon

that, alternative strategies were carried in the present work. The

ionic liquid matrix (ILM), produced from DHB and triethylamine,

furnished the greatest results regarding these matters, besides

allowing the high homogeneity of sample preparation, crucial

to automatic screening method. The analyte/ILM ratio was evalu-

ated as well. Best results include the lowest intensity ILM’s ions,

clear analyte ionization and ion preservation (low fragmentation).

These results can be related with the high vacuum stability of

ILM’s ions.[17]

The matrices with CTAB (hexadecyltrimethylammonium

bromide), a surfactant that suppresses the ions from matrix,

was analyzed at concentration ratios 10 000-100, but the ioniza-

tion suppression of several compounds could have beenobserved, what prevented its use. The ionic liquids were

prepared using equimolar proportions of 2,5-dihydroxybenzoic

acid (DHB), sinapinic acid, a-cyano-4-hydroxycinnamic acid matri-

ces and triethylamine.[18] The ionic liquid of DHB has shown the

best performance, and it was used in all HT-MSS experiments

(MALDI parameters: reflector mode, 1000 Hz laser frequency,

pulsed ion extraction of 100 ns, positive and negative modes).

The possible compounds of interest were selected and analyzed

by MS/MS using LIFT method to identify or suggest the

compounds. To support the accurate mass data and the fragmen-

tation information, the selected sample analyzed by MALDI was

also analyzed by LC-MS/MS. 100 mg of powdered leaves was

weighed in a glass vial and extracted in an ultrasonic bath for

10 min with 3 mL of MeOH:H2O (1:1) solution containing 2% of

acetic acid. Extract was filtered on a 0.45 mm cellulose acetate

membrane and submitted to HPLC analysis (C-18), by injection

of 20mL. The following elution gradient was employed, with a

flow rate of 3.0 mL min-1: solvent A (H2O with 2% of acetic acid),

and solvent B (MeCN with 2% of acetic acid. Elution profile

0–5 min, 10% B (isocratic), 5–8min, 10–40% B (linear gradient),

8–9 min, 40–10% B (linear reduction of gradient), and 9–11 min,

10% B (isocratic). The chromatogram proves strictosidine as

the major secondary metabolite (Fig. 2) and the possibility to

large-scale production.

The strategy reported here brings a new and effective way for

contemporary science to aggregate value to biodiversity, trans-

forming conservation and sustainable use into highly profitable

activities for forest inhabitants. With governmental willingness

and resources to establish extraction protocols with Associa-

tions/Cooperatives of Rainforest inhabitants, this strategy could

become a major source of income, converting local inhabitants

into the guardians of this natural treasure.

In view of recent discussion of Biodiversity Conservation, as

well as the recent forums of Rio + 20 meeting of global leaders,

we are presenting here yet another striking result of the BIOTA/ FAPESP, a long-term research Program on biodiversity science

previously reported by us[10] as a successful Brazilian experience

in gathering the advances from scientific knowledge with the

improvement of public policies on biodiversity conservation.

Bringing together scientists from different research areas

working under a common set of objectives, using standard proto-

cols, sharing data through electronic tools and brewing together

ideas to promote biodiversity conservation and sustainable use,

may be a good recipe to transform in reality CBD ’s targets. We

look forward to see the present strategy being useful for the

newly established Intergovernmental Platform on Biodiversity

and Ecosystem Services/IPBES[19] and that the approach outlined

here could also serve as a model for preservation throughout

the world.

Acknowledgements

Authors acknowledge FAPESP/BIOTA for grants 2003/12595-7,

2003/02176-7, 2009/54098-6, and 2010/50811-7. We thank the

students and technicians engaged in field work, in particular,

Belinello, R.; Padgurschi, M.C.G and Pereira, L.S. Permits CGEN/

IBAMA 093/2005 and COTEC/IF 41.065/2005.

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JMS letters

wileyonlinelibrary.com/journal/jms Copyright © 2012 John Wiley & Sons, Ltd. J. Mass Spectrom. 2012, 47 , 1482–1485

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JMS letters

J. Mass Spectrom. 2012, 47 , 1482–1485 Copyright © 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jms

http://www.biotaneotropica.org.br/v12n1/en/abstract?article+bn01812012012


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2012 Pavarini - Application of MALDI-MS Analysis of Rainforest

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