Aplicações de MALDI-MS na análise de peptídeos produzidos por cianobactérias
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Data
2017-06-30
Autores
Luizete, Milena Fontes [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Neste trabalho, a técnica Matrix Assisted Laser Dersoption/Ionization – Mass Spectrometry (MALDI-MS) foi utilizada em diferentes aplicações para a análise de peptídeos produzidos por cianobactérias. Esta técnica é extremamente rápida, possui alta resolução, requer pouquíssimo preparo de amostra e não permite que possíveis contaminantes presentes na amostra interfiram na análise. Por estes motivos MALDI- MS tem sido amplamente utilizada não somente na detecção de diferentes variantes de cianopeptídeos, mas também para quantificação das cianotoxinas. Este trabalho utilizou um sistema binário de matriz para a quantificação de microcistinas, utilizando as matrizes ácido α-ciano-4-hidroxicinamico e o ácido sinapínico (50/50, v/v) para obter uma mistura homogênea de matriz/analito, superando a baixa reprodutibilidade inerente da técnica MALDI-MS. Paralelamente, foi realizado o imageamento de espécies de cianobactérias de água doce cultivadas em meio sólido por MALDI-TOF-MS. Os resultados obtidos apresentaram novas perspectivas com relação à distribuição espacial dos peptídeos produzidos pelas espécies de cianobactérias estudadas, como a nodularina-R (m/z 825) e nodularina-[Har] (m/z 839) produzida pela espécie Nodularia harveyana PCC 7804, os peptídeos MC-LR (m/z 995) produzidos pela espécie Microcystis aeruginosa PCC 7820, e o sideróforo anaquelina (m/z 761.3) produzidos pela espécie Anabaena Cylindrica PCC 7122. Além disto, amostras da floração de cianobactérias, que ocorre na Represa Salto Grande, localizada na cidade de Americana – SP, foram analisadas e nelas foram identificados onze cianopeptídeos de quatro classes diferentes, sendo quatro microcistinas (MC-LR, MC-YR, MC-Hil e MC-RR), quatro aeruginosinas (602, 298 A, 644 e 646), duas cianopeptolinas (972 e 986), e uma variante de microviridina (1707).
In this work, the technique Matrix Assisted Laser Dersoption/Ionization – Mass Spectrometry (MALDI-MS) were used in different application for analysis of peptides produces by cyanobacteria. This technique is extremely fast, has high resolution, requires very little sample preparation and does not allow any contaminants present in the sample to interfere with the analysis. For these reasons, MALDI-MS has been widely used not only in the detection of different variants of cyanopeptides, but also for the quantification of cyanotoxins. This work utilized a matrix binary system for the quantification of microcystins, using the α-cyano-4-hydroxynamic matrix and sinapinic acid (50/20, v/v) to obtain a homogeneous matrix/analyte mixture, overcoming the low reproducibility inherent to the MALDI-MS technique. Simultaneously, we performed the imaging of freshwater cyanobacteria cultivated in solid medium by MALDI-TOF-MS. The results obtained presented new perspectives regarding the spatial distribution of the peptides produced by the studied cyanobacteria species, for exemple the nodularin-R (m/z 825), nodularin-[Har] (m/z 839) for the species Nodularia harveyana PCC 7804, MC-LR peptides (m/z 995) for the species Microcystis aeruginosa PCC 7820, and the siderophore anaquelin (m/z 761.3) for the species Anabaena cylindrica PCC 7122. In addition, samples of cianobacteria’s bloom, present in Salto Grande Lagoon, located in Americana-SP, were analyzed and were identified some peptides, being four microcystins (MC-YR, MC-YR, MC-Hil and MC-RR), four aeruginosins (602, 298 A, 644 and 968), two cyanopeptolins (972 and 986), and one variant of microviridin(1707).
In this work, the technique Matrix Assisted Laser Dersoption/Ionization – Mass Spectrometry (MALDI-MS) were used in different application for analysis of peptides produces by cyanobacteria. This technique is extremely fast, has high resolution, requires very little sample preparation and does not allow any contaminants present in the sample to interfere with the analysis. For these reasons, MALDI-MS has been widely used not only in the detection of different variants of cyanopeptides, but also for the quantification of cyanotoxins. This work utilized a matrix binary system for the quantification of microcystins, using the α-cyano-4-hydroxynamic matrix and sinapinic acid (50/20, v/v) to obtain a homogeneous matrix/analyte mixture, overcoming the low reproducibility inherent to the MALDI-MS technique. Simultaneously, we performed the imaging of freshwater cyanobacteria cultivated in solid medium by MALDI-TOF-MS. The results obtained presented new perspectives regarding the spatial distribution of the peptides produced by the studied cyanobacteria species, for exemple the nodularin-R (m/z 825), nodularin-[Har] (m/z 839) for the species Nodularia harveyana PCC 7804, MC-LR peptides (m/z 995) for the species Microcystis aeruginosa PCC 7820, and the siderophore anaquelin (m/z 761.3) for the species Anabaena cylindrica PCC 7122. In addition, samples of cianobacteria’s bloom, present in Salto Grande Lagoon, located in Americana-SP, were analyzed and were identified some peptides, being four microcystins (MC-YR, MC-YR, MC-Hil and MC-RR), four aeruginosins (602, 298 A, 644 and 968), two cyanopeptolins (972 and 986), and one variant of microviridin(1707).
Descrição
Palavras-chave
MALDI-MS, Espectrometria de massas, Cianobactérias, Microcistinas, Cianopeptídeos