Localization of Secondary Metabolites in Marine Invertebrates: Contribution of MALDI MSI for the Study of Saponins in Cuvierian Tubules of H. forskali

BACKGROUND: Several species of sea cucumbers of the family Holothuriidae possess a particular mechanical defense system called the Cuvierian tubules (Ct). It is also a chemical defense system as triterpene glycosides (saponins) appear to be particularly concentrated in Ct. In the present study, the precise localization of saponins in the Ct of Holothuria forskali is investigated. Classical histochemical labeling using lectin was firstly performed but did not generate any conclusive results. Thus, MALDI mass spectrometry Imaging (MALDI-MSI) was directly applied and completed by statistical multivariate tests. A comparison between the tubules of relaxed and stressed animals was realized. RESULTS: These analyses allowed the detection of three groups of ions, corresponding to the isomeric saponins of the tubules. Saponins detected at m/z 1287 and 1303 were the most abundant and were apparently localized in the connective tissue of the tubules of both relaxed and stressed individuals. Saponins at m/z 1125 and 1141 were detected in lower amount and were present in tissues of relaxed animals. Finally, saponin ions at 1433, 1449, 1463 and 1479 were observed in some Ct of stressed holothuroids in the outer part of the connective tissue. The saponin group m/z 14xx seems therefore to be stress-specific and could originate from modifications of the saponins with m/z of 11xx. CONCLUSIONS: All the results taken together indicate a complex chemical defense mechanism with, for a single organ, different sets of saponins originating from different cell populations and presenting different responses to stress. The present study also reflects that MALDI-MSI is a valuable tool for chemical ecology studies in which specific chemical signalling molecules like allelochemicals or pheromones have to be tracked. This report represents one of the very first studies using these tools to provide a functional and ecological understanding of the role of natural products from marine invertebrates

Identification of Hypoxia-Regulated Proteins Using MALDI-Mass Spectrometry Imaging Combined with Quantitative Proteomics

Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed. 2014 American Chemical Societ

MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the in situ analysis of tissue sections. MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement and enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. In this review, we focus on the current state of the art of MALDI-IMS, describe basic technological developments for MALDI-IMS of animal and human tissues, and discuss some recent applications in basic research and in clinical settings

Multiple Changes in Peptide and Lipid Expression Associated with Regeneration in the Nervous System of the Medicinal Leech

peer reviewe

Ovarian cancer molecular pathology.

Peer reviewe

DEVELOPPEMENTS EN IMAGERIE PAR<br />SPECTROMETRIE DE MASSE ET APPLICATIONS<br />AUX MODELES INVERTEBRES

At the proteomics' time, the mass spectrometry appears to be a powerful tool to findand identify peptides and proteins from purified sample. A new era is now beginning, with theMALDI imaging, allowing both research, identification while conserving the localization ofbiomolecules like peptides, proteins and lipids in tissues. Crucial developments at the level ofthe sample preparations are in progress to improve the performances of this technology. Inthis context, we were interested first of all in the settling of new protocols adapted to directanalysis and Imaging mass spectrometry of small organisms in particular the leech Hirudomedicinalis. This model is particularly interesting for the study of nervous regeneration andwe perfomed studies on lipids which can be implied in this physiological process. The secondpoint is the study of the contributions of metal coating for mass spectrometry. First of all ametal evaporated on histological glass allows at the same time a correlation of themorphological information with the molecular images of IMS. The coating of the sample,allowed preventing the mass shifts towards the highest m/z, to obtain best MALDI spectrumand according to a more important reproducibility between 2 spectra, to produce MALDIimages with better qualities. Finally, a last part of the developments was dedicated to thepossibility of improving the image resolution by using a system allowing decreasing theirradiated area by the laser.A l'heure de la protéomique, la spectrométrie de masse s'est révélée un outil puissantpour la recherche et l'identification des biomolécules à partir d'échantillons purifiés. Unenouvelle ère s'ouvre, avec l'imagerie MALDI, permettant en plus la localisation debiomolécules telles que les peptides, les protéines ou les lipides au sein des tissus. Desdéveloppements cruciaux restent encore à réaliser pour améliorer les performances de cettetechnologie. Dans ce contexte, nous nous sommes tout d'abord intéressés à la mise au pointde nouveaux protocoles adaptés à l'analyse directe et l'imagerie par spectromètrie de massede petits organismes en particulier la sangsue Hirudo medicinalis. Ce modèle estparticulièrement intéressant du point de vue des phénomènes de régénération nerveuse et nousavons débuté des études sur les lipides pouvant y être impliqués. Le deuxième point abordéest l'étude des apports de la métallisation pour la spectromètrie de masse. Tout d'abord undépôt métallique sur des lames histologiques permet à la fois une corrélation des informationsmorphologiques obtenues en microscopie optique avec les images moléculaires d'IMS. Lamétallisation de l'échantillon quand à elle, a permis de supprimer les décalages de pics versles plus hauts rapports m/z, d'obtenir des spectres MALDI de meilleures qualités et grâce àune reproductibilité plus importante entre 2 spectres, de produire des images MALDI de plusgrandes qualités. Enfin, une partie des développements a été dédiée à la possibilitéd'améliorer la résolution de l'image grâce à l'utilisation d'un système permettant de diminuerla zone accessible au lase

MALDI mass spectrometry imaging of proteins exceeding 30 000 daltons

Background: Since its introduction 10 years ago by Caprioli and associates, MALDI mass spectrometry imaging has enabled spatial analysis of drugs, lipids, peptides, and polypeptides. In polypeptides, the detectable mass range is limited to small proteins with a mass less than 25 kDa. This is a limitation, as many proteins, including cytokines, growth factors, enzymes, and receptors have molecular weights, exceeding 25 kDa. In the present work, we report the development of a novel strategy to observe higher mass proteins up to 30 kDa. Material/Methods: We investigated the development of sample preparation methods based on hexafluoroisopropanol (1,1,1,3,3,3-hexaluoro-2-propanol) and 2,2,2-trifluoroethanol solvents for protein solubilization optimized for high-mass proteins. Results: We were, for the first time in mass spectrometry imaging, able to detect to proteins up to 70 kDa directly from tissue. These developments indicate future avenues by which the sensitivity of protein mass spectrometry imaging can be further improved. We applied these developments to ovarian cancer and demonstrate that protein are similar to that which can be obtained using 2D gel based analyses. Conclusions: Increasing the possibility of detecting proteins and high-mass proteins is key for developing direct tissue proteomics and especially any potential functional investigation. These data will open the door of a novel step in mass spectrometry imaging