Untargeted omics analyses aim to comprehensively characterize biomolecules within a biological system. Changes in the presence or quantity of these biomolecules can indicate important biological perturbations, such as those caused by disease. With current technological advancements, the entire genome can now be sequenced; however, in the burgeoning fields of lipidomics, only a subset of lipids can be identified. The recent emergence of high resolution tandem mass spectrometry (HR-MS/MS), in combination with ultra-high performance liquid chromatography, has resulted in an increased coverage of the lipidome. Nevertheless, identifications from MS/MS are generally limited by the number of precursors which can be selected for fragmentation during chromatographic elution. Therefore, we developed the software IE-Omics to automate iterative exclusion (IE), where selected precursors using data-dependent topN analyses are excluded in sequential injections. In each sequential injection, unique precursors are fragmented until HR-MS/MS spectra of all ions above a user-defined intensity threshold are acquired. IE-Omics was applied to lipidomic analyses in Red Cross plasma and substantia nigra tissue. Coverage of the lipidome was drastically improved using IE. When applying IE-Omics to Red Cross plasma and substantia nigra lipid extracts in positive ion mode, 69 % and 40 % more molecular identifications were obtained, respectively. In addition, applying IE-Omics to a lipidomics workflow increased the coverage of trace species, including odd-chained and short-chained diacylglycerides and oxidized lipid species. By increasing the coverage of the lipidome, applying IE to a lipidomics workflow increases the probability of finding biomarkers and provides additional information for determining etiology of disease.

Preparation of tissue for matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) generally involves embedding the tissue followed by freezing and cryosectioning, usually between 5-25 μm thick, depending on the tissue type and the analyte(s) of interest. The brain is approximately 60% fat; it therefore lacks rigidity and poses structural preservation challenges during sample preparation. Histological sample preparation procedures are generally transferable to MALDI-MSI; however, there are various limitations. Optimal cutting temperature compound (OCT) is commonly used to embed and mount fixed tissue onto the chuck inside the cryostat during cryosectioning. However, OCT contains potential interferences that are detrimental to MALDI-MSI, whilst fixation is undesirable for the analysis of some analytes either due to extraction or chemical modification (i.e. polar metabolites). Therefore a method for both fixed and fresh tissue compatible with MALDI-MSI and histology is desirable to increase the breadth of analyte(s), maintain the topographies of the brain and provide rigidity to the fragile tissue whilst eliminating background interference. The method we introduce uses precast gelatin-based molds in which a whole mouse brain is embedded, flash frozen and cryosectioned in preparation for mass spectrometry imaging (MSI).

Microglia are the resident immune effector cells of the central nervous system. They account for approximately 10-15% of all cells found in the brain and spinal cord, acting as macrophages, sensing and engaging in phagocytosis to eliminate toxic proteins. Microglia are dynamic and can change their morphology in response to cues from their milieu. Parkinson's disease is a neurodegenerative disease, associated with reactive gliosis, neuroinflammation, and oxidative stress. It is thought that Parkinson's disease is caused by the accumulation of abnormally folded alpha-synuclein protein, accompanied by persistent neuroinflammation, oxidative stress, and subsequent neuronal injury/death. There is evidence in the literature for mitochondrial dysfunction in Parkinson's disease as well as fatty acid beta-oxidation, involving l-carnitine. Here we investigate l-carnitine in the context of microglial activation, suggesting a potential new strategy of supplementation for PD patients. Preliminary results from our studies suggest that the treatment of activated microglia with the endogenous antioxidant l-carnitine can reverse the effects of detrimental neuroinflammation in vitro.

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Parkinson's disease (PD) is a neurodegenerative disorder resulting from the loss of dopaminergic neurons of the substantia nigra as well as degeneration of motor and nonmotor basal ganglia circuitries. Typically known for classical motor deficits (tremor, rigidity, bradykinesia), early stages of the disease are associated with a large nonmotor component (depression, anxiety, apathy, etc.). Currently, there are no definitive biomarkers of PD, and the measurement of dopamine metabolites does not allow for detection of prodromal PD nor does it aid in long-term monitoring of disease progression. Given that PD is increasingly recognized as complex and heterogeneous, involving several neurotransmitters and proteins, it is of importance that we advance interdisciplinary studies to further our knowledge of the molecular and cellular pathways that are affected in PD. This approach will possibly yield useful biomarkers for early diagnosis and may assist in the development of disease-modifying therapies. Here, we discuss preanalytical factors associated with metabolomics studies, summarize current mass spectrometric methodologies used to evaluate the metabolic signature of PD, and provide future perspectives of the rapidly developing field of MS in the context of PD.

Liquid-microjunction surface sampling (LMJ-SS) is an ambient ionization technique based on the continuous flow of solvent using an in situ microextraction device in which solvent moves through the probe, drawing in the analytes in preparation for ionization using an electrospray ionization source. However, unlike traditional mass spectrometry (MS) techniques, it operates under ambient pressure and requires no sample preparation, thereby making it ideal for rapid sampling of thicker tissue sections for electrophysiological and other neuroscientific research studies. Studies interrogating neural synapses, or a specific neural circuit, typically employ thick, ex vivo tissue sections maintained under near-physiological conditions to preserve tissue viability and maintain the neural networks. Deep brain stimulation (DBS) is a surgical procedure used to treat the neurological symptoms that are associated with certain neurodegenerative and neuropsychiatric diseases. Parkinson's disease (PD) is a neurological disorder which is commonly treated with DBS therapy. PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta portion of the brain. Here, we demonstrate that the LMJ-SS methodology can provide a platform for ex vivo analysis of the brain during electrical stimulation, such as DBS. We employ LMJ-SS in the ex vivo analysis of mouse brain tissue for monitoring dopamine during electrical stimulation of the striatum region. The mouse brain tissue was sectioned fresh post sacrifice and maintained in artificial cerebrospinal fluid to create near-physiological conditions before direct sampling using LMJ-SS. A selection of metabolites, including time-sensitive metabolites involved in energy regulation in the brain, were identified using standards, and the mass spectral database mzCloud was used to assess the feasibility of the methodology. Thereafter, the intensity of m/z 154 corresponding to protonated dopamine was monitored before and after electrical stimulation of the striatum region, showing an increase in signal directly following a stimulation event. Dopamine is the key neurotransmitter implicated in PD, and although electrochemical detectors have shown such increases in dopamine post-DBS, this is the first study to do so using MS methodologies.

Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta of the brain, as well as the degeneration of motor and nonmotor circuitries. The cause of neuronal death is currently unknown, although chronic neuroinflammation, aggregated α-synuclein, mitochondrial dysfunction, and oxidative stress have all been implicated. Gliosis has been shown to exacerbate neuroinflammation via secretion of proinflammatory cytokines, and there is a subsequent infiltration of T lymphocytes (T-cells), into the brain of PD patients. Using liquid chromatography–high-resolution mass spectrometry (LC–HRMS), we have observed metabolomic changes in stool samples, thought to be associated with the potential disease-modifying effect of immunotherapy administered to transgenic Parkinsonian (A53T) mice. Significant elevations (p < 0.05) in metabolites associated with immune response (taurine, histamine, and its methylated product, 3-methylhistamine) are identified as being higher in the mice undergoing immunotherapy. Furthermore, a reduction in triacylglycerol (TG) and diacylglycerol (DG) expressions in stool following immunotherapy suggests a regulation of lipid breakdown or biosynthesis with the vaccine. These “omics” markers (among others reported in this article) along with weight gain and increased life expectancy suggest that immunotherapy is positively modifying the disease state.

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