Host-Guest Chemistry for Simultaneous Imaging of Endogenous Alkali Metals and Metabolites with Mass Spectrometry.

Sodium and potassium are biological alkali metal ions that are essential for the physiological processes of cells and organisms. In combination with small-molecule metabolite information, disturbances in sodium and potassium tissue distributions can provide a further understanding of the biological processes in diseases. However, methods using mass spectrometry are generally tailored toward either elemental or molecular detection, which limits simultaneous quantitative mass spectrometry imaging of alkali metal ions and molecular ions. Here, we provide a new method by including crown ether molecules in the solvent for nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) that combines host-guest chemistry targeting sodium and potassium ions and quantitative imaging of endogenous lipids and metabolites. After evaluation and optimization, the method was applied to an ischemic stroke model, which has highly dynamic tissue sodium and potassium concentrations, and we report 2 times relative increase in the detected sodium concentration in the ischemic region compared to healthy tissue. Further, in the same experiment, we showed the accumulation and depletion of lipids, neurotransmitters, and amino acids using relative quantitation with internal standards spiked in the nano-DESI solvent. Overall, we demonstrate a new method that with a simple modification in liquid extraction MSI techniques using host-guest chemistry provides the added dimension of alkali metal ion imaging to provide unique insights into biological processes.

CpG preconditioning reduces accumulation of lysophosphatidylcholine in ischemic brain tissue after middle cerebral artery occlusion.

Ischemic stroke is one of the major causes of death and permanent disability in the world. However, the molecular mechanisms surrounding tissue damage are complex and further studies are needed to gain insights necessary for development of treatment. Prophylactic treatment by administration of cytosine-guanine (CpG) oligodeoxynucleotides has been shown to provide neuroprotection against anticipated ischemic injury. CpG binds to Toll-like receptor 9 (TLR9) causing initialization of an inflammatory response that limits visible ischemic damages upon subsequent stroke. Here, we use nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging (MSI) to characterize molecular effects of CpG preconditioning prior to middle cerebral artery occlusion (MCAO) and reperfusion. By doping the nano-DESI solvent with appropriate internal standards, we can study and compare distributions of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) in the ischemic hemisphere of the brain despite the large changes in alkali metal abundances. Our results show that CpG preconditioning not only reduces the infarct size but it also decreases the degradation of PC and accumulation of LPC species, which indicates reduced cell membrane breakdown and overall ischemic damage. Our findings show that molecular mechanisms of PC degradation are intact despite CpG preconditioning but that these are limited due to the initialized inflammatory response.

Biodegradation of phenol by Chlamydomonas reinhardtii.

The data presented in this particular study demonstrate that the biodegradation of phenol by Chlamydomonas reinhardtii is a dynamic bioenergetic process mainly affected by the production of catechol and the presence of a growth-promoting substrate in the culture medium. The study focused on the regulation of the bioenergetic equilibrium resulting from production of catechol after phenol oxidation. Catechol was identified by HPLC-UV and HPLC-ESI-MS/MS. Growth measurements revealed that phenol is a growth-limiting substrate for microalgal cultures. The Chlamydomonas cells proceed to phenol biodegradation because they require carbon reserves for maintenance of homeostasis. In the presence of acetic acid (a growth-promoting carbon source), the amount of catechol detected in the culture medium was negligible; apparently, acetic acid provides microalgae with sufficient energy reserves to further biodegrade catechol. It has been shown that when microalgae do not have sufficient energy reserves, a significant amount of catechol is released into the culture medium. Chlamydomonas reinhardtii acts as a versatile bioenergetic machine by regulating its metabolism under each particular set of growth conditions, in order to achieve an optimal balance between growth, homeostasis maintenance and biodegradation of phenol. The novel findings of this study reveal a paradigm showing how microalgal metabolic versatility can be used in the bioremediation of the environment and in potential large-scale applications.

Investigating the Uptake of Arsenate by Chlamydomonas reinhardtii Cells and its Effect on their Lipid Profile using Single Cell ICP-MS and Easy Ambient Sonic-Spray Ionization-MS.

The complementary use of single cell atomic mass spectrometry (MS) and ambient molecular MS allowed for the in-depth study of arsenate uptake by Chlamydomonas reinhardtii cells and of the effect this toxic metalloid species has on their lipid profile. Compared to conventional inductively coupled plasma mass spectrometry (ICP-MS) analysis, in which case hundreds of thousands of cells are digested and then analyzed, it is demonstrated that single cell (SC) ICP-MS provides uptake data that are potentially of greater biological relevance. This includes the arsenic mass distribution within the cell population, which fits to a log-normal probability function, the most frequently contained arsenic mass within the cells (1.5-1.8 fg As per cell), and the mean arsenic uptake value (ranging from 2.7 to 4.1 fg As per cell for the three arsenate incubation concentrations, that is, 15, 22.5, and 30 µg As per mL) derived from the log-normal arsenic mass distribution within the cell population. The SC approach also allows for differentiating the arsenic present in and/or adsorbed on the cells, from the arsenic present in the extracellular solution, in a single analysis. In a similar fashion, ambient molecular MS in the form of desorption easy ambient sonic spray ionization (EASI) -MS was used to rapidly profile cell membrane lipids from cells spotted directly on a glass slide. EASI-MS analysis revealed that cells grown in the presence of increasing concentrations of arsenate exhibited changes in the degree of saturation of their membrane lipids, as was observed by the increasing intensity ratio of lipids with less unsaturated acyl chains to the same type of lipids with more unsaturated fatty acid chains. Thus, indicating "homeoviscous adaptation" of extraplastidial and thylakoid cell membranes, induced by the presence of arsenate.

Determination of chlorate, perchlorate and bromate anions in water samples by microbore reversed-phase liquid chromatography coupled to sonic-spray ionization mass spectrometry.

RATIONALE: Sonic-spray ionization mass spectrometry (SSI-MS) has recently been shown to provide similar mass spectra to those generated by electrospray ionization mass spectrometry for a wide range of compounds, i.e. from small inorganic species to peptides, proteins and numerous other biomolecules. However, limited information about this new ionization technique, such as sensitivity, limit of detection and quantification accuracy, has been reported. In particular, its coupling to liquid chromatography needs further development and assessment, along with the introduction of a broad range of applications. METHODS: A high-efficiency glass pneumatic nebulizer, used for decades for sample introduction in atomic spectrometry, was used for the SSI-MS analysis of chlorate (ClO3- ), perchlorate (ClO4- ) and bromate (BrO3- ) anions, following their separation using reversed-phase microbore high-performance liquid chromatography and tandem mass spectrometry (MS/MS) operated in selected reaction monitoring mode. RESULTS: The developed and optimized microbore HPLC/SSI-MS/MS technique exhibited low limits of detection: 5.3 ng L-1 for chlorate, 10 ng L-1 for perchlorate and 33.7 ng L-1 for bromate, and provided reliable and accurate measurements of chlorate concentrations in water samples as demonstrated when comparing it with Ion Chromatography-Conductivity Detection (IC-CD), the benchmark technique for ion quantitation. CONCLUSIONS: This is the first time that the use of HPLC/SSI-MS/MS has been reported for the detection and quantitation of chlorate, perchlorate and bromate in water samples. In addition, the exceptionally low LODs achieved using SSI render the technique competitive with the established and dominating electrospray ionization technique. Here, we have demonstrated that a commercially available high-efficiency glass pneumatic nebulizer can also be used, without any further modification, as an efficient gas-phase ion source. Copyright © 2017 John Wiley & Sons, Ltd.

Identification and Imaging of Prostaglandin Isomers Utilizing MS3 Product Ions and Silver Cationization.

Prostaglandins (PGs) are important lipid mediators involved in physiological processes, such as inflammation and pregnancy. The pleiotropic effects of the PG isomers and their differential expression from cell types impose the necessity for studying individual isomers locally in tissue to understand the molecular mechanisms. Currently, mass spectrometry (MS)-based analytical workflows for determining the PG isomers typically require homogenization of the sample and a separation method, which results in a loss of spatial information. Here, we describe a method exploiting the cationization of PGs with silver ions for enhanced sensitivity and tandem MS to distinguish the biologically relevant PG isomers PGE2, PGD2, and Δ12-PGD2. The developed method utilizes characteristic product ions in MS3 for training prediction models and is compatible with direct infusion approaches. We discuss insights into the fragmentation pathways of Ag+ cationized PGs during collision-induced dissociation and demonstrate the high accuracy and robustness of the model to predict isomeric compositions of PGs. The developed method is applied to mass spectrometry imaging (MSI) of mouse uterus implantation sites using silver-doped pneumatically assisted nanospray desorption electrospray ionization and indicates localization to the antimesometrial pole and the luminal epithelium of all isomers with different abundances. Overall, we demonstrate, for the first time, isomeric imaging of major PG isomers with a simple method that is compatible with liquid-based extraction MSI methods.

Matrix Effects Free Imaging of Thin Tissue Sections Using Pneumatically Assisted Nano-DESI MSI.

Mass spectrometry imaging has the potential to reveal important molecular interaction in morphological regions in tissue. However, the simultaneous ionization of the continuously altered and complex chemistry in each pixel can introduce artifacts that result in skewed molecular distributions in the compiled ion images. These artifacts are known as matrix effects. Mass spectrometry imaging using nanospray desorption electrospray ionization (nano-DESI MSI) enables the elimination of matrix effects by doping the nano-DESI solvent with internal standards. Carefully selected internal standards ionize similarly and simultaneously with the extracted analytes from thin tissue sections, and the matrix effects are eliminated through a robust data normalization method. Herein we describe the setup and use of pneumatically assisted (PA) nano-DESI MSI with standards doped in the solvent for elimination of matrix effects in ion images.

Ischemic Stroke Causes Disruptions in the Carnitine Shuttle System.

Gaining a deep understanding of the molecular mechanisms underlying ischemic stroke is necessary to develop treatment alternatives. Ischemic stroke is known to cause a cellular energy imbalance when glucose supply is deprived, enhancing the role for energy production via ß-oxidation where acylcarnitines are essential for the transportation of fatty acids into the mitochondria. Although traditional bulk analysis methods enable sensitive detection of acylcarnitines, they do not provide information on their abundances in various tissue regions. However, with quantitative mass spectrometry imaging the detected concentrations and spatial distributions of endogenous molecules can be readily obtained in an unbiased way. Here, we use pneumatically assisted nanospray desorption electrospray ionization mass spectrometry imaging (PA nano-DESI MSI) doped with internal standards to study the distributions of acylcarnitines in mouse brain affected by stroke. The internal standards enable quantitative imaging and annotation of endogenous acylcarnitines is achieved by studying fragmentation patterns. We report a significant accumulation of long-chain acylcarnitines due to ischemia in brain tissue of the middle cerebral artery occlusion (MCAO) stroke model. Further, we estimate activities of carnitine transporting enzymes and demonstrate disruptions in the carnitine shuttle system that affects the ß-oxidation in the mitochondria. Our results show the importance for quantitative monitoring of metabolite distributions in distinct tissue regions to understand cell compensation mechanisms involved in handling damage caused by stroke.

The effect of nitrogen starvation on membrane lipids of Synechocystis sp. PCC 6803 investigated by using easy ambient sonic-spray ionization mass spectrometry.

BACKGROUND: Glycerolipids are important components of membranes in cyanobacteria that possess vital roles in biological processes. The effect of nitrogen deprivation on membrane lipids of Synechocystis sp. PCC 6803 lipids has not been previously examined. METHODS: Easy ambient sonic-spray ionization mass spectrometry (EASI-MS) was used for the analysis of Synechocystis 6803 cells with minimal sample preparation, providing rapid qualitative and relative quantitative information on the lipid content of their membranes. RESULTS: Changes in the degree of unsaturation of membrane lipids were observed for cells grown under normal conditions during different growth phases. This physiological remodeling was disrupted when nitrogen was withdrawn from the cultivation medium. However, this disruption was reversed when the cells were resuspended in normal N-containing medium. Mass spectrometric data were supported by examination of cells by electron microscopy. CONCLUSIONS: EASI-MS was applied for the first time in the analysis of Synechocystis 6803 cells grown under nitrogen deprived conditions and was found to be a powerful technique operating in a high-throughput manner for the rapid lipid profiling of cells at different growth stages and under different growth conditions. GENERAL SIGNIFICANCE: The effect of nitrogen deprivation on membrane lipids of Synechocystis 6803 cells was revealed using an ambient ionization technique which enabled high-throughput cell analysis with minimal sample preparation. The results obtained have the potential to be used in future studies to decipher the involvement of enzymes in the observed lipid profile changes.