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Solvent separating secondary metabolites directly from biosynthetic tissue for surface-assisted laser desorption ionisation mass spectrometry.

Rudd D, Benkendorff K, Voelcker NH - Mar Drugs (2015)

Bottom Line: Water, ethanol, chloroform and hexane selectively extracted a range of choline esters, brominated indoles and lipids from Dicathais orbita hypobranchial tissue imprints.These compounds could be quantified on the nanostructured surfaces by comparison to standard curves generated from the pure compounds.Surface-assisted MS could have broad utility for detecting a broad range of secondary metabolites in complex marine tissue samples.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences, Faculty of Science and Engineering, Flinders University of South Australia, PO Box 2100, Adelaide, SA 5001, Australia. david.rudd@flinders.edu.au.

ABSTRACT
Marine bioactive metabolites are often heterogeneously expressed in tissues both spatially and over time. Therefore, traditional solvent extraction methods benefit from an understanding of the in situ sites of biosynthesis and storage to deal with heterogeneity and maximize yield. Recently, surface-assisted mass spectrometry (MS) methods namely nanostructure-assisted laser desorption ionisation (NALDI) and desorption ionisation on porous silicon (DIOS) surfaces have been developed to enable the direct detection of low molecular weight metabolites. Since direct tissue NALDI-MS or DIOS-MS produce complex spectra due to the wide variety of other metabolites and fragments present in the low mass range, we report here the use of "on surface" solvent separation directly from mollusc tissue onto nanostructured surfaces for MS analysis, as a mechanism for simplifying data annotation and detecting possible artefacts from compound delocalization during the preparative steps. Water, ethanol, chloroform and hexane selectively extracted a range of choline esters, brominated indoles and lipids from Dicathais orbita hypobranchial tissue imprints. These compounds could be quantified on the nanostructured surfaces by comparison to standard curves generated from the pure compounds. Surface-assisted MS could have broad utility for detecting a broad range of secondary metabolites in complex marine tissue samples.

No MeSH data available.


Related in: MedlinePlus

MSI detection region after EtOH separation of secondary metabolites on a DIOS surface. 15 μm thick tissue sections were imprinted onto the nanostructured surface then washed by gently pipetting solvent over the imprint area, onto a clean section of the surface (-----), where the solvent was then allowed to evaporate before LDI-MS; (a) scan of tissue on DIOS; (b) MSI of murexine 224 m/z; (c) MSI of tyrindoxyl sulfate 340 m/z; (d) MSI of tyrindoleninone 256 m/z; (e) MSI of Tyrian purple 421 m/z and (f) example an histological tissue section of the hypobranchial region stained with haematoxylin and eosin.
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marinedrugs-13-01410-f002: MSI detection region after EtOH separation of secondary metabolites on a DIOS surface. 15 μm thick tissue sections were imprinted onto the nanostructured surface then washed by gently pipetting solvent over the imprint area, onto a clean section of the surface (-----), where the solvent was then allowed to evaporate before LDI-MS; (a) scan of tissue on DIOS; (b) MSI of murexine 224 m/z; (c) MSI of tyrindoxyl sulfate 340 m/z; (d) MSI of tyrindoleninone 256 m/z; (e) MSI of Tyrian purple 421 m/z and (f) example an histological tissue section of the hypobranchial region stained with haematoxylin and eosin.

Mentions: Recently, we applied NALDI-MSI and DIOS-MSI to detect the spatial distribution of mollusc secondary metabolites via a tissue imprinting approach [19]. This approach allowed us to map the distribution of Tyrian purple and its precursors, but the elucidation of some spatially interesting spectra became challenging due to the complexity of spectral signals from the imprinted heterogeneous tissue samples. Spectral data from fragments of larger more labile molecules can be found within the low mass region, making identification of known secondary metabolites difficult and the interpretation of unknown or unexpected secondary metabolites complex. In order to improve secondary metabolite detection and elucidation, a simple chromatographic separation scheme was devised to selectively extract metabolites directly from the tissue on to the nanostructured surface for analysis. Direct separation onto the nanostructured surfaces can be done from frozen sections and under nitrogen gas (Figure 1), reducing enzymatic changes and atmospheric oxidative degradation, while maintaining the benefits of the nanostructured surfaces. Since LDI-MS analysis is also conducted in a high vacuum environment without light, oxidative and photo catalytic degradation affecting less stable secondary metabolites is reduced. The solvent wash area adjacent to the tissue (Figure 2) can subsequently be used to concentrate particular subsets of compounds according to their solubility, thus facilitating identification based on simpler spectra and structural features, e.g. polarity, halogenation. Furthermore, this technique can enable quantification of natural products from tissue imprints by generating standard concentration curves from the purified compounds on the adjacent nanostructured surfaces for comparison of the LDI-MS signal intensity.


Solvent separating secondary metabolites directly from biosynthetic tissue for surface-assisted laser desorption ionisation mass spectrometry.

Rudd D, Benkendorff K, Voelcker NH - Mar Drugs (2015)

MSI detection region after EtOH separation of secondary metabolites on a DIOS surface. 15 μm thick tissue sections were imprinted onto the nanostructured surface then washed by gently pipetting solvent over the imprint area, onto a clean section of the surface (-----), where the solvent was then allowed to evaporate before LDI-MS; (a) scan of tissue on DIOS; (b) MSI of murexine 224 m/z; (c) MSI of tyrindoxyl sulfate 340 m/z; (d) MSI of tyrindoleninone 256 m/z; (e) MSI of Tyrian purple 421 m/z and (f) example an histological tissue section of the hypobranchial region stained with haematoxylin and eosin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4377991&req=5

marinedrugs-13-01410-f002: MSI detection region after EtOH separation of secondary metabolites on a DIOS surface. 15 μm thick tissue sections were imprinted onto the nanostructured surface then washed by gently pipetting solvent over the imprint area, onto a clean section of the surface (-----), where the solvent was then allowed to evaporate before LDI-MS; (a) scan of tissue on DIOS; (b) MSI of murexine 224 m/z; (c) MSI of tyrindoxyl sulfate 340 m/z; (d) MSI of tyrindoleninone 256 m/z; (e) MSI of Tyrian purple 421 m/z and (f) example an histological tissue section of the hypobranchial region stained with haematoxylin and eosin.
Mentions: Recently, we applied NALDI-MSI and DIOS-MSI to detect the spatial distribution of mollusc secondary metabolites via a tissue imprinting approach [19]. This approach allowed us to map the distribution of Tyrian purple and its precursors, but the elucidation of some spatially interesting spectra became challenging due to the complexity of spectral signals from the imprinted heterogeneous tissue samples. Spectral data from fragments of larger more labile molecules can be found within the low mass region, making identification of known secondary metabolites difficult and the interpretation of unknown or unexpected secondary metabolites complex. In order to improve secondary metabolite detection and elucidation, a simple chromatographic separation scheme was devised to selectively extract metabolites directly from the tissue on to the nanostructured surface for analysis. Direct separation onto the nanostructured surfaces can be done from frozen sections and under nitrogen gas (Figure 1), reducing enzymatic changes and atmospheric oxidative degradation, while maintaining the benefits of the nanostructured surfaces. Since LDI-MS analysis is also conducted in a high vacuum environment without light, oxidative and photo catalytic degradation affecting less stable secondary metabolites is reduced. The solvent wash area adjacent to the tissue (Figure 2) can subsequently be used to concentrate particular subsets of compounds according to their solubility, thus facilitating identification based on simpler spectra and structural features, e.g. polarity, halogenation. Furthermore, this technique can enable quantification of natural products from tissue imprints by generating standard concentration curves from the purified compounds on the adjacent nanostructured surfaces for comparison of the LDI-MS signal intensity.

Bottom Line: Water, ethanol, chloroform and hexane selectively extracted a range of choline esters, brominated indoles and lipids from Dicathais orbita hypobranchial tissue imprints.These compounds could be quantified on the nanostructured surfaces by comparison to standard curves generated from the pure compounds.Surface-assisted MS could have broad utility for detecting a broad range of secondary metabolites in complex marine tissue samples.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences, Faculty of Science and Engineering, Flinders University of South Australia, PO Box 2100, Adelaide, SA 5001, Australia. david.rudd@flinders.edu.au.

ABSTRACT
Marine bioactive metabolites are often heterogeneously expressed in tissues both spatially and over time. Therefore, traditional solvent extraction methods benefit from an understanding of the in situ sites of biosynthesis and storage to deal with heterogeneity and maximize yield. Recently, surface-assisted mass spectrometry (MS) methods namely nanostructure-assisted laser desorption ionisation (NALDI) and desorption ionisation on porous silicon (DIOS) surfaces have been developed to enable the direct detection of low molecular weight metabolites. Since direct tissue NALDI-MS or DIOS-MS produce complex spectra due to the wide variety of other metabolites and fragments present in the low mass range, we report here the use of "on surface" solvent separation directly from mollusc tissue onto nanostructured surfaces for MS analysis, as a mechanism for simplifying data annotation and detecting possible artefacts from compound delocalization during the preparative steps. Water, ethanol, chloroform and hexane selectively extracted a range of choline esters, brominated indoles and lipids from Dicathais orbita hypobranchial tissue imprints. These compounds could be quantified on the nanostructured surfaces by comparison to standard curves generated from the pure compounds. Surface-assisted MS could have broad utility for detecting a broad range of secondary metabolites in complex marine tissue samples.

No MeSH data available.


Related in: MedlinePlus