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

Application of MALDI-MS analysis of Rainforest chemodiversity: a keystone for biodiversity conservation 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 has demonstrated 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 prots, triggering a perverse logic that prot 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 protable. 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 oristic[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) ionizationbased 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 understand ecophysiological traits of leaves, and MALDI-MS was an alternative to identify alkaloids as one of the nitrogen sink. Regarding the following main points: (1) analysis of plant attributed nitrogen xation and (2) protable policies for biodiversity conservation, 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 occurrence of alkaloids and botanical identication. Classical chemical procedures were also applied to validate the results (Dragendorff, Mayer and Wagner assays). In this letter, we provide the rst 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 small molecules (< 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 approach, we can screen up to 200 samples within a 3 h time-frame.

* Correspondence to: Norberto P. Lopes, Ncleo Pesquisas em Produtos Naturais e Sintticos, Departamento de Fsica e Qumica, Faculdade de Cincias Farmacuticas de Ribeiro Preto, Universidade de So Paulo, Zipcode 14040-903, Ribeiro Preto, SP, Brazil. E-mail: npelopes@fcfrp.usp.br * Carlos A. Joly, Plant Biology Department, Biology Institute, State University of Campinas/UNICAMP, POBox 6109, ZIPCode 13083-970 Campinas, SP, Brazil. E-mail: cjoly@unicamp.br

Present Address: Instituto de Pesquisas Jardim Botnico do Rio de Janeiro, Diretoria de Pesquisas. 22460-030- Rio de Janeiro, RJ Brazil

a Ncleo Pesquisas em Produtos Naturais e Sintticos, Departamento de Fsica e Qumica, Faculdade de Cincias Farmacuticas de Ribeiro Preto, Universidade de So Paulo, Zipcode 14040-903, Ribeiro Preto, SP, Brazil b Lychnoora, Pesquisa e Desenvolvimento em Produtos Naturais, Universidade de So Paulo, Zipcode 14040-903, Ribeiro Preto, SP, Brazil c Departamento de Farmcia-Bioqumica, Centro de Cincias Biolgicas e da Sade, Universidade Federal de Mato Grosso do Sul, POBox 549, ZIPCode 79070-900, Campo Grande, MS, Brazil d Programa de Ps-Graduao em Biologia Vegetal, Universidade Estadual de Campinas, Zipcode 13083-970, Campinas, SP, Brazil e Ncleo de Pesquisa em Fisiologia e Bioqumica, Instituto de Botnica/SMA, POBox 68042, ZIPCode 04045-972, So Paulo, SP, Brazil f Plant Biology Department, Biology Institute, State University of Campinas/UNICAMP, 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 Ministrios, Braslia, Brazil h Instituto de Qumica de Araraquara, Departamento de Qumica de Qumica Orgnica, NuBBE, Universidade Estadual Paulista, POBox 355, ZIPCode 14800-900, Araraquara, SP, Brazil

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JMS letters This is a 192-fold increase in throughput, and therefore it can be efciently 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

x104 5 4

311.2186 304.1826

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0.5 185.1257
308.1819 311.2142 185.0934 230.9351 390.2162 529.2097607.1731 193.0442 230.9716

359.2084

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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 CTAB matrix, D: with the ionic liquid matrix produced from DHB and triethylamine, E: MS/MS spectrum of strictosidine. All the illustrated spectra were done in positive 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).

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JMS letters are used to conrm 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 oristic 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 dene the economical perspectives. Only 5% of the selected species showed the presence of alkaloids, suggesting a low N-xation through secondary metabolite storage. For economically 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] Strictosidine 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 prot on yielding leaves compounds by single solvent-recrystallization methods and prot by supplying Research Spin-off companies with the yielded compounds. For this, rst, 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, different matrices were tested, and the best matrix was ionic liquid (Fig. 2). The matrix plays a main role in the ionization and desorption 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 evaluated as well. Best results include the lowest intensity ILMs ions, clear analyte ionization and ion preservation (low fragmentation). These results can be related with the high vacuum stability of ILMs 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 ionization suppression of several compounds could have been observed, 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 matrices and triethylamine.[18] The ionic liquid of DHB has shown the best performance, and it was used in all HT-MSS experiments (MALDI parameters: reector 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 fragmentation 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 ltered 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 ow 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 prole 05 min, 10% B (isocratic), 58 min, 1040% B (linear gradient), 89 min, 4010% B (linear reduction of gradient), and 911 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, transforming conservation and sustainable use into highly protable activities for forest inhabitants. With governmental willingness and resources to establish extraction protocols with Associations/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 scientic 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 protocols, 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 CBDs 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 eld 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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