Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations

AbstractS100A8 and S100A9 are two proinflammatory molecules belonging to the S100 family of calcium-binding proteins. Common to all S100 proteins S100A8 and S100A9 form non-covalently associated complexes which have been shown to exhibit different functional properties. Besides dimerization, recent research is focused on the importance of higher oligomeric structures of S100 proteins induced by bivalent cations. While S100A8/S100A9-heterodimers are formed in the absence of calcium, tetramerization is strictly calcium-dependent. Heterodimer formation is not a simple process and our biophysical analyses (FRET, ESI-MS) demonstrate that simply mixing both subunits is not sufficient to induce complex formation. Steps of denaturation/renaturation are necessary for the recombinant complex to show identical biophysical properties as S100A8/S100A9 obtained from granulocytes. In addition to calcium both proteins are able to bind zinc with high affinity. Here we demonstrate for the first time by different biophysical methods (MALDI-MS, ESI-MS, fluorescence spectroscopy) that zinc-binding, like calcium, induces (S100A8/S100A9)2-tetramers. Using mass spectrometric investigations we demonstrate that zinc triggers the formation of (S100A8/S100A9)2-tetramers by zinc-specific binding sites rather than by interactions with calcium-specific EF-hands. The zinc-induced tetramer is structurally very similar to the calcium-induced tetramer. Thus, like calcium, zinc acts as a regulatory factor in S100A8/S100A9-dependent signaling pathways

Spatial Mapping of Lipids at Cellular Resolution in Embryos of Cotton

Advances in mass spectrometry (MS) have made comprehensive lipidomics analysis of complex tissues relatively commonplace. These compositional analyses, although able to resolve hundreds of molecular species of lipids in single extracts, lose the original cellular context from which these lipids are derived. Recently, high-resolution MS of individual lipid droplets from seed tissues indicated organelle-to-organelle variation in lipid composition, suggesting that heterogeneity of lipid distributions at the cellular level may be prevalent. Here, we employed matrix-assisted laser desorption/ionization–MS imaging (MALDI-MSI) approaches to visualize lipid species directly in seed tissues of upland cotton (Gossypium hirsutum). MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phosphatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applications for this chemical visualization approach. We conclude that comprehensive lipidomics images generated by MALDI-MSI report accurate, relative amounts of lipid species in plant tissues and reveal previously unseen differences in spatial distributions providing for a new level of understanding in cellular biochemistry

In vivo distribution of tiotropium in a rodent model utilizing AP-SMALDI mass spectrometry imaging

Background: Matrix-assisted laser desorption/ionization mass spectrometry imaging has proved to be a powerful tool in localization of endogenous molecules in thin tissue sections. Distributions of drug compounds following administration in experimental animal models can be spatially detected and superimposed upon histological images thereby identifying the tissue compartments involved in drug uptake, transfer and metabolism. Methods: A new instrumental setup consisting of a Thermo Scientific Q Exactive mass spectrometer and an AP SMALDI10 ion source was used for imaging data acquisition at 10-µm lateral resolution to provide details of the distribution of a bronchodilator, tiotropium, in a rodent model. Results: The extracted ion maps of tiotropium showed higher signal intensities in bronchioles compared to lung parenchyma, while the drug was absent in lymphoid nodes and blood vessels. The enhanced mass spectrometric image quality provided refined spatial distribution in comparison with data obtained on a Thermo Scientific MALDI LTQ Orbitrap XL mass spectrometer obtained at 30, 80 and 150 µm lateral resolution. Conclusion: The high spatial resolution images of administrated tiotropium in rat lung obtained by the AP SMALDI10 ion source have readily improved the localization of this drug, highlighting cellular level distributions from the airways to the parenchyma

Visualizing Neurotransmitters and Metabolites in the Central Nervous System by High Resolution and High Accuracy Mass Spectrometric Imaging

The spatial localization and molecular distribution of metabolites and neurotransmitters within biological organisms is of tremendous interest to neuroscientists. In comparison to conventional imaging techniques such as immunohistochemistry, matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) has demonstrated its unique advantage by directly localizing the distribution of a wide range of biomolecules simultaneously from a tissue specimen. Although MALDI-MSI of metabolites and neurotransmitters is hindered by numerous matrix-derived peaks, high-resolution and high-accuracy mass spectrometers (HRMS) allow differentiation of endogenous analytes from matrix peaks, unambiguously obtaining biomolecular distributions. In this study, we present MSI of metabolites and neurotransmitters in rodent and crustacean central nervous systems acquired on HRMS. Results were compared with those obtained from a medium-resolution mass spectrometer (MRMS), tandem time-of-flight instrument, to demonstrate the power and unique advantages of HRMSI and reveal how this new tool would benefit molecular imaging applications in neuroscience

Spatial Mapping of Lipids at Cellular Resolution in Embryos of Cotton

Article on the spatial mapping of lipids at cellular resolution in embryos of cotton