Filomena Petruzziello

High Identification Rates of Endogenous Neuropeptides from Mouse Brain

Mass spectrometry-based neuropeptidomics is one of the most powerful approaches for identification of endogenous neuropeptides in the brain. Until now, however, the identification rate of neuropeptides in neuropeptidomics is relatively low and this severely restricts insights into their biological function. In the present study, we developed a high accuracy mass spectrometry-based approach to enhance the identification rates of neuropeptides from brain tissue. Our integrated approach used mixing on column for loading aqueous and organic extracts to reduce the loss of peptides during sample treatment and used charge state-directed tandem mass spectrometry to increase the number of peptides subjected to high mass accuracy fragmentation. This approach allowed 206 peptides on average to be identified from a single mouse brain sample that was prepared using 15 μL of solutions per 1 mg of tissue. In total, we identified more than 500 endogenous peptides from mouse hypothalamus and whole brain samples. Our identification rate is about two to four times higher compared to previously reported studies conducted on mice or other species. The hydrophobic peptides, such as neuropeptide Y and galanin, could be presented and detected with hydrophilic peptides in the same LC-MS run, allowing a high coverage of peptide characterization over an organism. This will advance our understanding of the roles of diverse peptides and their links in the brain functions.

Analysis of multiple quaternary ammonium compounds in the brain using tandem capillary column separation and high resolution mass spectrometric detection.

Endogenous quaternary ammonium compounds are involved in various physiological processes in the central nervous system. In the present study, eleven quaternary ammonium compounds, including acetylcholine, choline, carnitine, acetylcarnitine and seven other acylcarnitines of low polarity, were analyzed from brain extracts using a two dimension capillary liquid chromatography-Fourier transform mass spectrometry method. To deal with their large difference in hydrophobicities, tandem coupling between reversed phase and hydrophilic interaction chromatography columns was used to separate all the targeted quaternary ammonium compounds. Using high accuracy mass spectrometry in selected ion monitoring mode, all the compounds could be detected from each brain sample with high selectivity. The developed method was applied for the relative quantification of these quaternary ammonium compounds in three different brain regions of tree shrews: prefrontal cortex, striatum, and hippocampus. The comparative analysis showed that quaternary ammonium compounds were differentially distributed across the three brain areas. The analytical method proved to be highly sensitive and reliable for simultaneous determination of all the targeted analytes from brain samples.

Extensive Characterization of Tupaia belangeri Neuropeptidome Using an Integrated Mass Spectrometric Approach

Neuropeptidomics is used to characterize endogenous peptides in the brain of tree shrews (Tupaia belangeri). Tree shrews are small animals similar to rodents in size but close relatives of primates, and are excellent models for brain research. Currently, tree shrews have no complete proteome information available on which direct database search can be allowed for neuropeptide identification. To increase the capability in the identification of neuropeptides in tree shrews, we developed an integrated mass spectrometry (MS)-based approach that combines methods including data-dependent, directed, and targeted liquid chromatography (LC)-Fourier transform (FT)-tandem MS (MS/MS) analysis, database construction, de novo sequencing, precursor protein search, and homology analysis. Using this integrated approach, we identified 107 endogenous peptides that have sequences identical or similar to those from other mammalian species. High accuracy MS and tandem MS information, with BLAST analysis and chromatographic characteristics were used to confirm the sequences of all the identified peptides. Interestingly, further sequence homology analysis demonstrated that tree shrew peptides have a significantly higher degree of homology to equivalent sequences in humans than those in mice or rats, consistent with the close phylogenetic relationship between tree shrews and primates. Our results provide the first extensive characterization of the peptidome in tree shrews, which now permits characterization of their function in nervous and endocrine system. As the approach developed fully used the conservative properties of neuropeptides in evolution and the advantage of high accuracy MS, it can be portable for identification of neuropeptides in other species for which the fully sequenced genomes or proteomes are not available.

Chronic nicotine treatment impacts the regulation of opioid and non-opioid peptides in the rat dorsal striatum

The chronic use of nicotine, the main psychoactive ingredient of tobacco smoking, alters diverse physiological processes and consequently generates physical dependence. To understand the impact of chronic nicotine on neuropeptides, which are potential molecules associated with dependence, we conducted qualitative and quantitative neuropeptidomics on the rat dorsal striatum, an important brain region implicated in the preoccupation/craving phase of drug dependence. We used extensive LC-FT-MS/MS analyses for neuropeptide identification and LC-FT-MS in conjunction with stable isotope addition for relative quantification. The treatment with chronic nicotine for 3 months led to moderate changes in the levels of endogenous dorsal striatum peptides. Five enkephalin opioid peptides were up-regulated, although no change was observed for dynorphin peptides. Specially, nicotine altered levels of nine non-opioid peptides derived from precursors, including somatostatin and cerebellin, which potentially modulate neurotransmitter release and energy metabolism. This broad but selective impact on the multiple peptidergic systems suggests that apart from the opioid peptides, several other peptidergic systems are involved in the preoccupation/craving phase of drug dependence. Our finding permits future evaluation of the neurochemical circuits modulated by chronic nicotine exposure and provides a number of novel molecules that could serve as potential therapeutic targets for treating drug dependence.

Neuropeptide alterations in the tree shrew hypothalamus during volatile anesthesia

Neuropeptides are critical signaling molecules, involved in the regulation of diverse physiological processes including energy metabolism, pain perception and brain cognitive state. Prolonged general anesthesia has an impact on many of these processes, but the regulation of peptides by general anesthetics is poorly understood. In this study, we present an in-depth characterization of the hypothalamic neuropeptides of the tree shrew during volatile isoflurane/nitrous oxide anesthesia administered accompanying a neurosurgical procedure. Using a predicted-peptide database and hybrid spectral analysis, we first identified 85 peptides from the tree shrew hypothalamus. Differential analysis was then performed between control and experimental group animals. The levels of 12 hypothalamic peptides were up-regulated following prolonged general anesthesia. Our study revealed for the first time that several neuropeptides, including alpha-neoendorphin and somatostatin-14, were altered during general anesthesia. Our study broadens the scope for the involvement of neuropeptides in volatile anesthesia regulation, opening the possibility for investigating the associated regulatory mechanisms.

Quantitative Profiling of Endogenous Fat-Soluble Vitamins and Carotenoids in Human Plasma Using an Improved UHPSFC-ESI-MS Interface

Analytical solutions enabling the quantification of circulating levels of liposoluble micronutrients such as vitamins and carotenoids are currently limited to either single or a reduced panel of analytes. The requirement to use multiple approaches hampers the investigation of the biological variability on a large number of samples in a time and cost efficient manner. With the goal to develop high-throughput and robust quantitative methods for the profiling of micronutrients in human plasma, we introduce a novel, validated workflow for the determination of 14 fat-soluble vitamins and carotenoids in a single run. Automated supported liquid extraction was optimized and implemented to simultaneously parallelize 48 samples in 1 h, and the analytes were measured using ultrahigh-performance supercritical fluid chromatography coupled to tandem mass spectrometry in less than 8 min. An improved mass spectrometry interface hardware was built up to minimize the post-decompression volume and to allow better control of the chromatographic effluent density on its route toward and into the ion source. In addition, a specific make-up solvent condition was developed to ensure both analytes and matrix constituents solubility after mobile phase decompression. The optimized interface resulted in improved spray plume stability and conserved matrix compounds solubility leading to enhanced hyphenation robustness while ensuring both suitable analytical repeatability and improved the detection sensitivity. The overall developed methodology gives recoveries within 85-115%, as well as within and between-day coefficient of variation of 2 and 14%, respectively.

Altered neurochemical levels in the rat brain following chronic nicotine treatment.

Converging evidence shows that neurochemical systems are crucial mediators of nicotine dependence. Our present study evaluates the effect of 3-month chronic nicotine treatment on the levels of multiple quaternary ammonium compounds as well as glutamate and gamma aminobutyric acid in the rat prefrontal cortex, dorsal striatum and hypothalamus. We observed a marked decrease of acetylcholine levels in the dorsal striatum (22.88%, p<0.01), reflecting the impact of chronic nicotine in local interneuron circuits. We found decreases of carnitine in the dorsal striatum and prefrontal cortex (19.44%, p<0.01; 13.58%, p<0.01, respectively), but robust enhancements of carnitine in the hypothalamus (26.59%, p<0.01), which may reflect the alterations in food and water intake during chronic nicotine treatment. Finally, we identified an increase of prefrontal cortex glutamate levels (8.05%, p<0.05), supporting previous studies suggesting enhanced prefrontal activity during chronic drug use. Our study shows that quaternary ammonium compounds are regulated in a highly brain region specific manner during chronic nicotine treatment, and provides novel insights into neurochemical regulation during nicotine use.

Broad characterization of endogenous peptides in the tree shrew visual system

Endogenous neuropeptides, acting as neurotransmitters or hormones in the brain, carry out important functions including neural plasticity, metabolism and angiogenesis. Previous neuropeptide studies have focused on peptide-rich brain regions such as the striatum or hypothalamus. Here we present an investigation of peptides in the visual system, composed of brain regions that are generally less rich in peptides, with the aim of providing the first broad overview of peptides involved in mammalian visual functions. We target three important parts of the visual system: the primary visual cortex (V1), lateral geniculate nucleus (LGN) and superior colliculus (SC). Our study is performed in the tree shrew, a close relative of primates. Using a combination of data dependent acquisition and targeted LC-MS/MS based neuropeptidomics; we identified a total of 52 peptides from the tree shrew visual system. A total of 26 peptides, for example GAV and neuropeptide K were identified in the visual system for the first time. Out of the total 52 peptides, 27 peptides with high signal-to-noise-ratio (>10) in extracted ion chromatograms (EIC) were subjected to label-free quantitation. We observed generally lower abundance of peptides in the LGN compared to V1 and SC. Consistently, a number of individual peptides showed high abundance in V1 (such as neuropeptide Y or somatostatin 28) and in SC (such as somatostatin 28 AA1-12). This study provides the first in-depth characterization of peptides in the mammalian visual system. These findings now permit the investigation of neuropeptide-regulated mechanisms of visual perception.

High-Efficiency Recognition and Identification of Disulfide Bonded Peptides in Rat Neuropeptidome Using Targeted Electron Transfer Dissociation Tandem Mass Spectrometry

The main goal of the present study is to develop a method to recognize and identify endogenous intrachain disulfide bonded peptide, which are rarely sequenced in current peptidomics studies. In order to achieve highly efficient detection of these peptides in a neuropeptidome analysis, we alkylated the peptides, mined the raw mass spectrometry data, and then recognized the candidates of untreated disulfide bonded peptides from unalkylated peptide extracts. After removing more than 90% features, targeted electron transfer dissociation fragmentation was performed for detecting and fragmenting disulfide bonded peptides, and even most of them were present in low abundance in the original sample. Diverse endogenous disulfide bonded peptides were then detected and sequenced, opening up new perspectives for comprehensively understanding the response of a neuropeptidome.

Separation and identification of mouse brain tissue microproteins using top‐down method with high resolution nanocapillary liquid chromatography mass spectrometry

Microproteins and endogenous peptides in the brain contain important substances that have critical roles in diverse biological processes, contributing to signal transduction and intercellular signaling. However, variability in their physical or chemical characteristics, such as molecule size, hydrophobicity, and charge states, complicate the simultaneous analysis of these compounds, although this would be highly beneficial for the field of neuroscience research. Here, we present a top‐down analytical method for simultaneous analysis of microproteins and endogenous peptides using high‐resolution nanocapillary LC‐MS/MS. This method is detergent‐free and digestion‐free, which allows for extracting and preserving intact microproteins and peptides for direct LC‐MS analysis. Both higher energy collision dissociation and electron‐transfer dissociation fragmentations were used in the LC‐MS analysis to increase the identification rate, and bioinformatics tools ProteinGoggle and PEAKS Studio software were utilized for database search. In total, we identified 471 microproteins containing 736 proteoforms, including brain‐derived neurotrophic factor and a number of fibroblast growth factors. In addition, we identified 599 peptides containing 151 known or potential neuropeptides such as somatostatin‐28 and neuropeptide Y. Our approach bridges the gap for the characterization of brain microproteins and peptides, which permits quantification of a diversity of signaling molecules for biomarker discovery or therapy diagnosis in the future.