Orthogonal label and label-free dual pretreatment for targeted profiling of neurotransmitters in enteric nervous system
Graphical abstract
Introduction
The immune system in the gastrointestinal tract is continuously exposed to numerous microbiota, pathogens, and food-derived antigens. Unlike other peripheral organs, the gastrointestinal tract has a specific nervous system defined as enteric nervous system (ENS), which has critical roles in regulating the intestinal barrier function and interaction with immune systems [1,2]. Notably, in enteric reflection pathways, the neurons utilize a broad range of chemical neurotransmitters (NTs) and their metabolites that signal through an even more extensive range of receptors [3]. Previous researchers have observed that NTs orchestrate intestinal immune. The principal NT acetylcholine could drive the intestinal cholinergic anti-inflammatory pathway through the function in efferent vagal neurons [4]. Tryptophan and its metabolites, including tryptamine and kynurenine, could activate the aryl hydrocarbon receptor (AHR) in lymphoid tissues and promote Treg cell development [5,6].
Nowadays, targeted metabolic profiling becomes an essential tool in prior studies to predict outcomes and provide insights into the pathophysiology of disease processes in clinical research [7,8]. Given such crucial immunological significance, global profiling of NT metabolome may serve as a clinical indicator of the diagnosis and treatment of immune-inflammation diseases, thereby offering novel insights into the in-depth mechanism by which endogenous metabolites interact with the neuro-immune system. Moreover, NT metabolome may be useful in clinical research for understanding neurodegenerative disease mechanisms through microbiome-gut-brain axis [9].
Compared with validated single biomarkers used in clinical practice, panel-based NT metabolome profiling potentially represents more biochemical pathways and biological disturbance under inflammation [10,11]. The NT metabolome consists of small molecule metabolites, including cholines, amines, amino acids, and their metabolites [9] (Fig. 1). From the angle of chemical structures, amine and phenol NTs could be detected by LC-MS/MS-based chemical labeling approach [12,13]. More recently, some highly polar NTs were successfully determined by LC-MS/MS with a hydrophilic interaction liquid chromatography (HILIC) column [14]. However, only partial coverage of the NT metabolome can be simultaneously quantified by any of these approaches. This is due to the lack of all-purpose labeling reagents for vast chemical group diversity and the ionization efficiency differences in positive and negative ion modes.
Previous studies have shown that sample pretreatment optimization could increase metabolite coverage [15,16]. We recently achieved significant enhancement in microbial metabolite detectability and much higher metabolome coverage by integrating biphasic extractions with chemical derivatizations [17]. Adhering to a divide-and-conquer concept where the NTs could be divided into different chemical groups (Fig. 2), an orthogonal dual pretreatment approach was proposed for the NT metabolome simultaneous determination. First, a high-performance dansyl-labeling method was applied for targeting amine and phenol NTs. Meanwhile, for targeted analyzing tertiary amine and choline NTs, the label-free aliquot was designed to combine with the dansyl-labeling aliquot in a single vial. The orthogonal design will allow us to prioritize the development efforts on labeling chemistry to target specific groups of metabolites and compensate for the deficiency of the rest compounds in a metabolome to guide future method optimization.
With this method, we can monitor the global changes of enteric NTs in serum and intestinal contents samples from a dextran sulphate sodium (DSS)-induced colitis and the intervention effects of a Chinese patent drug. Comprehensive analysis of NTs in the host and microbiome showed that amino acids and cholines shifted in response to inflammation, and highlighted the biological significance of tryptophan and phenylalanine metabolism. Our approach aims at increasing the clinical attention of NT metabolome changes in inflammatory bowel disease and the mechanism of how NT metabolome plays a crucial part in maintaining the neuro-immune system. We hope that combining the divide-and-conquer concept with orthogonal dual pretreatment makes it possible to quantify more metabolites in targeted metabolomics.
Section snippets
Chemicals and reagents
The authentic compounds, including 3-hydroxykynurenine, 3, 4-dihydroxyphenylacetic acid, dopamine, homovanillic acid, were purchased from Sigma Aldrich (St. Louis, MO). The other chemicals (listed in Table 1) were obtained from Aladdin (Shanghai, China). The internal standard (IS), 2-chloro-L-phenylalanine (2-Cl-phe), was also purchased from Aladdin (Shanghai, China). LC-MS grade acetonitrile (ACN), formic acid, ammonium formate, and dansyl chloride were purchased from Merck (Darmstadt,
UPLC-MS/MS method optimization for targeted NT metabolome profiling
Although much efforts have been made to the detection of NTs, comprehensive profiling of NT metabolome within a single LC−MS run remains challenging. In the preliminary experiment, we have compared the base peak ion chromatograms of highly polar small molecule metabolites in the intestinal contents before and after derivatization under positive ion mode. As shown in Figure S2a, the responses of most ions appeared in the latter part of chromatogram with lower intensity and poor separation. After
Conclusion
NT signaling abnormalities in the ENS can trigger inflammation in the intestine. The involvement of both the ENS and the immune system in symptoms of UC has been continuously unveiled, including the dysregulations of NT metabolome in various immune disorders. However, due to the lack of an appropriate quantitative method, few studies have comprehensively delineated the metabolic profile of such an important metabolic panel. Depending on the divide-and-conquer concept, the present research
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 81673568, 81773872 and 81973577). This research was also supported by Talents planning of six summit fields of Jiangsu Province (No. YY-057). We also greatly appreciate the financial supports from the priority academic program development of Jiangsu higher education institutions (PAPD).
References (43)
The enteric nervous system: normal functions and enteric neuropathies
Neurogastroenterology & Motil.
(2008)The enteric nervous system and neurogastroenterology
Nat. Rev. Gastroenterol. Hepatol.
(2012)- et al.
The enteric nervous system
Dev. Biol.
(2012) - et al.
Impaired acetylcholine release in the inflamed rat intestine is T cell independent
Am. J. Physiol.
(1992) - et al.
An interaction between kynurenine and the aryl hydrocarbon receptor can generate regulatory T cells
J. Immunol.
(2010) - et al.
An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor
Nature
(2011) - et al.
A targeted metabolomics approach for clinical diagnosis of inborn errors of metabolism
Anal. Chim. Acta
(2018) - et al.
Next-generation metabolic screening: targeted and untargeted metabolomics for the diagnosis of inborn errors of metabolism in individual patients
J. Inherit. Metab. Dis.
(2018) - et al.
Mass spectrometry-based metabolomics: targeting the crosstalk between gut microbiota and brain in neurodegenerative disorders
Mass Spectrom. Rev.
(2019) - et al.
Metabolomics toward personalized medicine
Mass Spectrom. Rev.
(2019)
Analytical pitfalls and challenges in clinical metabolomics
Bioanalysis
Quantitative analysis of neurochemical panel in rat brain and plasma by liquid chromatography-tandem mass spectrometry
Anal. Chem.
Determination of dansylated monoamine and amino acid neurotransmitters and their metabolites in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry
Anal. Biochem.
Quantification of endogenous neurotransmitters and related compounds by liquid chromatography coupled to tandem mass spectrometry
Talanta
In-vial dual extraction for direct LC-MS analysis of plasma for comprehensive and highly reproducible metabolic fingerprinting
Anal. Chem.
Differential 12C-/13C-isotope dansylation labeling and fast liquid chromatography/mass spectrometry for absolute and relative quantification of the metabolome
Anal. Chem.
Development and validation of a systematic platform for broad-scale profiling of microbial metabolites
Talanta
Ammonium fluoride-induced stabilization for anion attachment mass spectrometry: facilitating the pseudotargeted profiling of bile acids submetabolome
Anal. Chim. Acta
Chemically induced mouse models of acute and chronic intestinal inflammation
Nat. Protoc.
Neurotransmitter modulation by the gut microbiota
Brain Res.
Neurotransmitters: the critical modulators regulating gut-brain Axis
J. Cell. Physiol.
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