Correlative mass spectrometry imaging, applying time-of-flight secondary ion mass spectrometry and atmospheric pressure matrix-assisted laser desorption/ionization to a single tissue section.

RATIONALE: Mass spectrometry imaging (MSI) is a powerful tool for mapping the surface of a sample. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) offer complementary capabilities. Here, we present a workflow to apply both techniques to a single tissue section and combine the resulting data for the example of human colon cancer tissue. METHODS: Following cryo-sectioning, images were acquired using the high spatial resolution (1 µm pixel size) provided by TOF-SIMS. The same section was then coated with a para-nitroaniline matrix and images were acquired using AP-MALDI coupled to an Orbitrap mass spectrometer, offering high mass resolution, high mass accuracy and tandem mass spectrometry (MS/MS) capabilities. Datasets provided by both mass spectrometers were converted into the open and vendor-independent imzML file format and processed with the open-source software MSiReader. RESULTS: The TOF-SIMS and AP-MALDI mass spectra show strong signals of fatty acids, cholesterol, phosphatidylcholine and sphingomyelin. We showed a high correlation between the fatty acid ions detected with TOF-SIMS in negative ion mode and the phosphatidylcholine ions detected with AP-MALDI in positive ion mode using a similar setting for visualization. Histological staining on the same section allowed the identification of the anatomical structures and their correlation with the ion images. CONCLUSIONS: This multimodal approach using two MSI platforms shows an excellent complementarity for the localization and identification of lipids. The spatial resolution of both systems is at or close to cellular dimensions, and thus spatial correlation can only be obtained if the same tissue section is analyzed sequentially. Data processing based on imzML allows a real correlation of the imaging datasets provided by these two technologies and opens the way for a more complete molecular view of the anatomical structures of biological tissues.

Wildfire responses to abrupt climate change in North America.

It is widely accepted, based on data from the last few decades and on model simulations, that anthropogenic climate change will cause increased fire activity. However, less attention has been paid to the relationship between abrupt climate changes and heightened fire activity in the paleorecord. We use 35 charcoal and pollen records to assess how fire regimes in North America changed during the last glacial-interglacial transition (15 to 10 ka), a time of large and rapid climate changes. We also test the hypothesis that a comet impact initiated continental-scale wildfires at 12.9 ka; the data do not support this idea, nor are continent-wide fires indicated at any time during deglaciation. There are, however, clear links between large climate changes and fire activity. Biomass burning gradually increased from the glacial period to the beginning of the Younger Dryas. Although there are changes in biomass burning during the Younger Dryas, there is no systematic trend. There is a further increase in biomass burning after the Younger Dryas. Intervals of rapid climate change at 13.9, 13.2, and 11.7 ka are marked by large increases in fire activity. The timing of changes in fire is not coincident with changes in human population density or the timing of the extinction of the megafauna. Although these factors could have contributed to fire-regime changes at individual sites or at specific times, the charcoal data indicate an important role for climate, and particularly rapid climate change, in determining broad-scale levels of fire activity.

A layered structure in the organic envelopes of the prismatic layer of the shell of the pearl oyster Pinctada margaritifera (Mollusca, Bivalvia).

The organic interprismatic layers of the mollusc Pinctada margaritifera are studied using a variety of highly spatially-resolved techniques to establish their composition and structure. Our results show that both the interlamellar sheets of the nacre and interprismatic envelopes form layered structures. Additionally, these organic layers are neither homogeneous in composition, nor continuous in their structure. Both structures play a major role in the biomineralization process and act as a boundary between mineral units.

Transcription regulatory proteins in higher plants.

Novel structures and interactions for plant transcription factors have recently been reported. Factors that lack DNA-binding activity, exhibit putative triple-helix DNA-binding motifs, or possess two DNA-binding domains are among the interesting results. Advances in our understanding of protein-protein interactions between plant transcription factors are facilitating the design of new experimental strategies to further define their roles in regulatory pathways.

[Imaging mass spectrometry: a new tool for the analysis of skin biopsy. Application in Fabry's disease]. / Imagerie par spectrométrie de masse: un nouvel outil pour l'analyse de biopsies cutanées. Application à la maladie de Fabry.

The advent of innovative techniques in mass spectrometry, especially in the area of imaging, prompted us to evaluate two promising techniques: secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. For this purpose, sections of cutaneous biopsies from patients affected by Fabry's disease and control patients were analyzed. In the course of this disease, two physiological glycosphingolipids [globotriasylceramide (Gb3) and the galabiosylceramide (Ga2)] accumulate in certain tissues owing to a catabolism failure. The ability of these techniques to localize sites of accumulation in body tissues and their capacity to identify the accumulated lipid structures by mass spectra were evaluated. Results demonstrated that these two techniques provide complementary information:-secondary ion mass spectrometry enabled precise localization of areas of accumulation with lateral resolution in the micrometer range;-the signal obtained with matrix-assisted laser desorption/ionization mass spectrometry was high enough to identify these structures according to their molecular weight.

High mass and spatial resolution mass spectrometry imaging of Nicolas Poussin painting cross section by cluster TOF-SIMS.

The painting Rebecca and Eliezer at the Well, which hangs in the Fitzwilliam Museum, Cambridge, UK, is possibly one of the last figure painting executed by Nicolas Poussin at the very end of his life and is usually dated to the early 1660s. In this perspective special feature, Philippe Walter, Alain Brunelle and colleagues give new insights on the artist's working methods by a careful stateof-the-art imaging ToF-SIMS study of one sample taken on the edge of the painting. This approach allowed for the identification of the pigments used in the painting, their nature and components and those of the ground and preparatory layers, with the identification of the binder(s) and possible other additions of organic materials such as glue. This study paves the way to a wider use of ToF-SIMS for the analysis of ancient cultural heritage artefacts. Dr. Walter is the Director of the Molecular and Structural Archeology Laboratory (Université Pierre et Marie Curie, Paris, France). Dr. Brunelle is Head of the Mass Spectrometry Laboratory at the Institut de Chimie des Substances Naturelles (CNRS, Gif-sur-Yvette, France). Their long standing collaboration has led to several seminal publications on the analysis of ancient artefacts by mass spectrometry.

Determining residue-base interactions between AraC protein and araI DNA.

Depurination/depyrimidation binding-interference experiments (missing contact probing) identified specific candidate residue-base interactions lost by mutants of Escherichia coli L-arabinose operon regulatory protein, AraC, to one of its binding sites, araI. These candidates were then checked more rigorously by comparing the affinities of wild-type and alanine-substituted AraC protein to variants of araI with alterations in the candidate contacted positions. Residues 208 and 212 apparently contact DNA and support, but do not prove the existence of a helix-turn-helix structure in this region of AraC protein whereas contacts by mutants with alterations at positions 256, 257 and 261 which are within another potential helix-turn-helix region do not support the existence of such a structure there. The missing contacts displayed by three AraC mutants are found within two major groove regions of the DNA and are spaced 21 base-pairs apart in a pattern indicating a direct repeat orientation for the subunits of AraC.

The classification and discrimination of glass fragments using non destructive energy dispersive X-ray microfluorescence.

Frequency of analytical characteristics is best estimated on glass recovered at random. However, as such data were not available to us, we decided to use control windows for this estimation. In order to use such a database, one has to establish that the recovered fragment comes from a window. Therefore, elemental analysis was used both for classification and discrimination of glass fragments. Several articles have been published on the subject, but most methods alter the glass sample. The use of non destructive energy dispersive X-ray microfluorescence (microXRF) for the analysis of small glass fragments has been evaluated in this context. The refractive index (RI) has also been measured in order to evaluate the complementarity of techniques. Classification of fragments has been achieved using Fisher's linear discriminant analysis (LDA) and neural networks (NN). Discrimination was based on Hotelling's T2 test. Only pairs that were not differentiated by RI followed by the Welch test were studied. The results show that neural network and linear discriminant analysis using qualitative and semi-quantitative data establishes a classification of glass specimens with a high degree of reliability. For discrimination, 119 windows collected from crime scene were compared: using RI it was possible to distinguish 6892 pairs. Out of 129 remaining pairs, 112 were distinguished by microXRF.

Missing contact probing of DNA-protein interactions.

We have examined the positions of contact between lambda phage repressor protein and operator OR1 DNA by scanning populations of lightly depurinated or depyrimidated DNA for bases essential to or irrelevant to repressor binding. This global scanning technique delineates the apparent contact region between lambda repressor and operator and shows bases previously demonstrated or predicted to be contacted plus some additional bases. A mutant repressor, previously shown to contact DNA as wild-type repressor does with the exception of a missing contact to guanosine G4' [Hochschild, A. & Ptashne, M. (1986) Cell 44, 925-933], similarly failed to contact G4' when assayed by this method. Coupled with altering a test residue of a DNA-contacting protein to glycine or alanine so as to eliminate a specific contact, the method appears to provide an efficient means of scanning for specific residue-base contacts.

Altered DNA contacts made by a mutant AraC protein.

Mutant AraC proteins were selected for their ability to induce but not to repress, or their ability to repress but not to induce the araBAD operon. One such unusual mutant is able to bind to the araI site with an affinity only two to three-fold weaker than the wild type AraC protein, but the mutant protein was shown, both in crude extracts and when purified, to contact only two of the three major groove regions of the DNA that are contacted by the wild type protein.

252Cf-plasma desorption mass spectrometry of unmodified lipid A: fragmentation patterns and localization of fatty acids.

The fragmentation patterns of synthetic Escherichia coli-type lipid A in plasma desorption mass spectrometry (PDMS) in both negative- and positive-ion modes were determined. Negative-ion spectra gave signals for the main diphosphorylated (intact) molecular species in their native proportions. Intact and alkaline-treated lipid A in this mode gave, for the glucosamine I moiety, easily identified signals that have not been previously reported in PDMS. These spectra gave enough information to localize the fatty acids. The procedure was verified with relatively homogeneous lipids A prepared from Salmonella minnesota R595 and Neisseria meningitidis lipopolysaccharides, and then applied to the previously unstudied Yersinia entercolitica O:11,24 lipid A to obtain the localization of its fatty acids. The possibility of obtaining this much information from two negative-ion spectra was attributed to the method, described earlier, of preparing the samples. In the positive-ion mode, about half of the E. coli ions containing diglucosamine appeared as monodephosphorylated species and/or as Na adducts. The intact glucosamine II moiety and its fragment ions gave signals none of which were Na adducts. With lipids A prepared from S. minnesota, N. meningitidis, and Y. enterocolitica, similar fragmentation patterns were observed. For lipid A structure determination, the positive-ion mode could play a confirmatory role. The above results and some of those reported by others were compared.

Functional analysis of petunia floral homeotic MADS box gene pMADS1.

The petunia mutant green petal (gp, line PLV) shows a homeotic effect in one floral whorl, that is, the conversion of petal to sepal. We demonstrate that this mutant contains a chromosomal deletion, including the petunia MADS box gene pMADS1. Second whorl petal development in this null mutant can be restored with a CaMV 35S-pMADS1 transgene, demonstrating the essential role of pMADS1 in this process. Because gp (PLV) shows only a minor effect on stamen development, the homeotic effects of pMADS1 are different from those of B-type genes in Antirrhinum and Arabidopsis. Two other MADS box genes, pMADS2 and fbp1 (Angenent et al. 1992), require pMADS1 to maintain expression in the second whorl. However, in the absence of pMADS1 these two genes continue to be expressed in the third whorl. The functions assigned to pMADS1 are further supported by experiments in which we phenocopy gp by cosuppression of pMADS1 gene expression. The flowers, obtained through cosuppression and phenotype restoration, display different degrees of sepal to petal conversion. Analysis of these flowers indicate that pMADS1 controls growth under the zone of petal and stamen initiation, which causes the corolla tube and stamen filaments to emerge as a congenitally fused structure.

Characterization of two human flavin-containing monooxygenase (form 3) enzymes expressed in Escherichia coli as maltose binding protein fusions.

To examine the possibility for drug metabolism polymorphism, adult human flavin-containing monooxygenases (form 3) (EC 1.14.13.8) that differ at one amino acid were expressed in Escherichia coli as maltose binding protein fusions. The cDNA that was first reported during the cloning of adult human flavin-containing monooxygenase was designated the wild type lys158 enzyme. A second cDNA has been identified as a common polymorphism in some human populations and was designated the glu158 enzyme. The cDNA that encodes both enzymes was subcloned into a high yield protein fusion expression system, expressed, and the protein was partially purified by affinity chromatography and characterized for enzyme activity with selective functional substrate probes. N- and S-oxygenation activity of both enzymes was determined with 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine and methyl p-tolyl sulfide, respectively. It was found that expression of both lys158 and glu158 enzymes of the human flavin-containing monooxygenase form 3 as fusions with the maltose binding protein resulted in an enzyme that was soluble and greatly stabilized and had a reduced requirement for detergent during enzyme purification and during the assay for activity. Expression of the fusion proteins has allowed the preparation of stable and highly active enzyme at greater purity than was readily possible in the past. With the exception of the stability and solubility characteristics, the physical and chemical properties of lys158 and glu158 maltose binding fusion proteins of human flavin-containing monooxygenase form 3 variants resembled that of flavin-containing monooxygenase enzyme activity associated with human liver microsomes and enzyme isolated from a previous Escherichia coli expression system that lacked the protein fusion. Comparison of the catalytic activity of the two fusion proteins showed that while both forms were active, there were differences in their substrate specificities. Expression of the adult human flavin-containing monooxygenase form 3 as a maltose binding protein has allowed considerable advances over the previously reported cDNA-expressed enzyme systems and may provide the basis for examining the role of the flavin-containing monooxygenase in human xenobiotic or drug metabolism.

Inhibition of glutamate carboxypeptidase II by phosphonamidothionate derivatives of glutamic acid.

A limited series of N-thiophosphonyl-glutamates were found to be inhibitors of the prostate-specific membrane antigen (PSMA) form of glutamate carboxypeptidase II. Comparative inhibitory profiles of an analogous O-thiophosphonyl-2-hydroxyglutarate revealed that the amido-linkage of the N-thiophosphonyl-glutamate provides a significant enhancement of inhibitory potency presumably due to significant hydrogen-bonding interactions with acceptor groups in the active-site of PSMA resulting in tighter binding. An analogous N-phosphonyl-glutamate exhibited significantly greater inhibitory potency than the parent N-thiophosphonyl-glutamate indicating that the sulfur ligand of the N-thiophosphonyl-glutamates is responsible for less favorable active-site interactions than oxygen, potentially due to steric crowding from the longer P-S bond or as a result of active-site metal substitution of Co(II) for Zn(II) arising from assay conditions.