Céline Mériaux

Liquid ionic matrixes for MALDI mass spectrometry imaging of lipids

Lipids are a major component of cells and play a variety of roles in organisms. In general, they play a key role in the structural composition of membranes. Some lipids, such as sphingoglycolipids, however, are also mediators of different biological processes, including protein transport, regulation of cell growth, cellular morphogenesis, neuronal plasticity, and regulation of the immune response. With the advent of MALDI mass spectrometry imaging (MALDI MSI), lipids have begun to be intensively investigated by several groups. Here we present a novel development in the detection and study of lipids using an automatic microspotter coupled to specific liquid ionic matrixes based on a 2,5-DHB matrix (i.e., 2,5-DHB/ANI, 2,5-DHB/Pyr, and 2,5-DHB/3-AP). This development allows to decrease the time of the sample preparation by comparison with crystalline 2,5-DHB as matrix and was validated on human ovarian cancer biopsies to demonstrate its use as a precise procedure that is particularly useful for specific diagnoses.

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.

Automated querying and identification of novel peptides using MALDI mass spectrometric imaging.

MSI is a molecular imaging technique that allows for the generation of topographic 2D maps for various endogenous and some exogenous molecules without prior specification of the molecule. In this paper, we start with the premise that a region of interest (ROI) is given to us based on preselected morphological criteria. Given an ROI, we develop a pipeline, first to determine mass values with distinct expression signatures, localized to the ROI, and second to identify the peptides corresponding to these mass values. To identify spatially differentiated masses, we implement a statistic that allows us to estimate, for each spectral peak, the probability that it is over- or under-expressed within the ROI versus outside. To identify peptides corresponding to these masses, we apply LC-MS/MS to fragment endogenous (nonprotease digested) peptides. A novel pipeline based on constructing sequence tags de novo from both original and decharged spectra and a subsequent database search is used to identify peptides. As the MSI signal and the identified peptide are only related by a single mass value, we isolate the corresponding transcript and perform a second validation via in situ hybridization of the transcript. We tested our approach, MSI-Query, on a number of ROIs in the medicinal leech, Hirudo medicinalis, including the central nervous system (CNS). The Hirudo CNS is capable of regenerating itself after injury, thus forming an important model system for neuropeptide identification. The pipeline helps identify a number of novel peptides. Specifically, we identify a gene that we name HmIF4, which is a member of the intermediate filament family involved in neural development and a second novel, uncharacterized peptide. A third peptide, derived from the histone H2B, is also identified, in agreement with the previously suggested role of histone H2B in axon targeting.

AMASS: Algorithm for MSI Analysis by Semi-supervised Segmentation

Mass Spectrometric Imaging (MSI) is a molecular imaging technique that allows the generation of 2D ion density maps for a large complement of the active molecules present in cells and sectioned tissues. Automatic segmentation of such maps according to patterns of co-expression of individual molecules can be used for discovery of novel molecular signatures (molecules that are specifically expressed in particular spatial regions). However, current segmentation techniques are biased toward the discovery of higher abundance molecules and large segments; they allow limited opportunity for user interaction, and validation is usually performed by similarity to known anatomical features. We describe here a novel method, AMASS (Algorithm for MSI Analysis by Semi-supervised Segmentation). AMASS relies on the discriminating power of a molecular signal instead of its intensity as a key feature, uses an internal consistency measure for validation, and allows significant user interaction and supervision as options. An automated segmentation of entire leech embryo data images resulted in segmentation domains congruent with many known organs, including heart, CNS ganglia, nephridia, nephridiopores, and lateral and ventral regions, each with a distinct molecular signature. Likewise, segmentation of a rat brain MSI slice data set yielded known brain features and provided interesting examples of co-expression between distinct brain regions. AMASS represents a new approach for the discovery of peptide masses with distinct spatial features of expression. Software source code and installation and usage guide are available at http://bix.ucsd.edu/AMASS/ .

Ionic matrices pre-spotted matrix-assisted laser desorption/ionization plates for patient maker following in course of treatment, drug titration, and MALDI mass spectrometry imaging.

In the current study, we compared plastic matrix-assisted laser desorption/ionization (MALDI) plates pre-spotted with different solid ionic matrices. Data reflect that after 3 months of storage, the standards were oxidized in α-cyano-4-hydroxycinnamic acid (HCCA) whether or not in HCCA/3-acetylpyridine (3APY) and HCCA/aniline, and certain peptides, such as ubiquitin, were not detected using the HCCA matrix, whereas they were detected in pre-spotted ionic matrices. Application in peptidomics of these MALDI matrices pre-spotted plates (after 3 months of storage) with ovarian cyst fluid showed less intense signals with HCCA than with solid ionic matrices. We show that these pre-spotted ionic matrices plates can be used for relative drug quantification, high mass protein detection, and MALDI mass spectrometry imaging.

Multiple changes in peptide and lipid expression associated with regeneration in the nervous system of the medicinal leech

Background The adult medicinal leech central nervous system (CNS) is capable of regenerating specific synaptic circuitry after a mechanical lesion, displaying evidence of anatomical repair within a few days and functional recovery within a few weeks. In the present work, spatiotemporal changes in molecular distributions during this phenomenon are explored. Moreover, the hypothesis that neural regeneration involves some molecular factors initially employed during embryonic neural development is tested. Results Imaging mass spectrometry coupled to peptidomic and lipidomic methodologies allowed the selection of molecules whose spatiotemporal pattern of expression was of potential interest. The identification of peptides was aided by comparing MS/MS spectra obtained for the peptidome extracted from embryonic and adult tissues to leech transcriptome and genome databases. Through the parallel use of a classical lipidomic approach and secondary ion mass spectrometry, specific lipids, including cannabinoids, gangliosides and several other types, were detected in adult ganglia following mechanical damage to connected nerves. These observations motivated a search for possible effects of cannabinoids on neurite outgrowth. Exposing nervous tissues to Transient Receptor Potential Vanilloid (TRPV) receptor agonists resulted in enhanced neurite outgrowth from a cut nerve, while exposure to antagonists blocked such outgrowth. Conclusion The experiments on the regenerating adult leech CNS reported here provide direct evidence of increased titers of proteins that are thought to play important roles in early stages of neural development. Our data further suggest that endocannabinoids also play key roles in CNS regeneration, mediated through the activation of leech TRPVs, as a thorough search of leech genome databases failed to reveal any leech orthologs of the mammalian cannabinoid receptors but revealed putative TRPVs. In sum, our observations identify a number of lipids and proteins that may contribute to different aspects of the complex phenomenon of leech nerve regeneration, establishing an important base for future functional assays.

Human temporal lobe epilepsy analyses by tissue proteomics.

Although there are many types of epilepsy, temporal lobe epilepsy (TLE) is probably in humans the most common and most often studied. TLE represents 40% of the total epilepsy form of the disease and is difficult to treat. Despite a wealth of descriptive data obtained from the disease history of patients, the EEG recording, imaging techniques, and histological studies, the epileptogenic process remains poorly understood. However, it is unlikely that a single factor or a single mechanism can cause many changes associated with this neuropathological phenomenon. MALDI mass spectrometry imaging (MSI) coupled to protein identification, because of its ability to study a wide range of molecules, appears to be suitable for the preparation of molecular profiles in TLE. Seven neuropeptides have been have been identified in Dental gyrus regions of the hippocampus in relation with TLE pathology. Shot‐gun studies taking into account gender influence have been performed. Tissue microextraction from control (10) toward 10 TLE patients have been analyzed after trypsin digestion followed by separation on nanoLC coupled to LTQ orbitrap. From the shot‐gun analyses, results confirmed the presence of specific neuropeptides precursors and receptors in TLE patients as well as proteins involved in axons regeneration including neurotrophins, ECM proteins, cell surface proteins, membrane proteins, G‐proteins, cytoskeleton proteins and tumor suppressors. Among the tumor suppressors identified, the Leucine‐rich glioma inactivated 1 (LGI1) protein was found. LGI1 gene recently been demonstrated being implicated in heritability of TLE. We have also demonstrate the presence a complete profile of tumor suppressors in TLE patients, 7 have been identified. Refining this analysis taken into account the gender influence in both control and in TLE reflected the presence of specific proteins between male and female and thus mechanisms in pathology development could be completely different.

Proteomic analysis of the spatio-temporal based molecular kinetics of acute spinal cord injury identifies a time- and segment-specific window for effective tissue repair

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target.

Brain Proteomics: Sample Preparation Techniques for the Analysis of Rat Brain Samples Using Mass Spectrometry

Rat brain proteomics is progressing gradually from gel-based approaches to gel-free systems and the emerging technique of MALDI imaging is making its mark on direct analysis of proteins in the brain sections. In this protocol chapter, we provide the details on the sample preparation methods for brain proteins analysis by mass spectrometry. Sample preparation methods include first grinding the brain and/or its regions in liquid nitrogen, followed by extracting proteins efficiently with various extraction buffers. The two protocols described here, are the modified TCA/acetone extraction method and extraction using Tris-buffered saline with addition of Tween 20. The soluble proteins obtained from the powdered brain samples can be directly analyzed after digestion with trypsin by gel-free approach to enhance the proteome coverage. Finally, we present a detailed protocol for protein identification in brain sections using the emerging technology of MALDI imaging.

MALDI Imaging Mass Spectrometry for Investigating the Brain

Dynamic properties of the nervous system can now be investigated through mass spectrometry technologies. Generally, the application of these powerful techniques requires the destruction of the specimen under study/examination, but recent technological advances have made it possible to directly analyze tissue sections and perform 2-D or 3-D molecular ions mapping. We review the increasing application of matrix-assisted laser desorption/ionization (MALDI) imaging to the analysis of molecular distributions of proteins and peptides in nervous tissues of both invertebrates and vertebrates, focusing in particular on recent studies of neurodegenerative diseases, and early efforts to implement assays of neuronal development.