Comprehensive Profiling of Fecal Metabolome of Mice by Integrated Chemical Isotope Labeling-Mass Spectrometry Analysis

Abstract

Gut microbiota plays important roles in the host health. The host and symbiotic gut microbiota coproduce a large number of metabolites during the metabolism of food and xenobiotics. The analysis of fecal metabolites can provide a noninvasive manner to study the outcome of the host-gut microbiota interaction. Herein, we reported the comprehensive profiling of fecal metabolome of mice by an integrated chemical isotope labeling combined with liquid chromatography-mass spectrometry (CIL-LC-MS) analysis. The metabolites are categorized into several submetabolomes based on the functional moieties (i.e., carboxyl, carbonyl, amine, and thiol) and then analysis of the individual submetabolome was performed. The combined data from the submetabolome form the metabolome with relatively high coverage. To this end, we synthesized stable isotope labeling reagents to label metabolites with different groups, including carboxyl, carbonyl, amine, and thiol groups. We detected 2302 potential metabolites, among which, 1388 could be positively or putatively identified in feces of mice. We then further confirmed 308 metabolites based on our established library of chemically labeled standards and tandem mass spectrometry analysis. With the identified metabolites in feces of mice, we established mice fecal metabolome database, which can be used to readily identify metabolites from feces of mice. Furthermore, we discovered 211 fecal metabolites exhibited significant difference between Alzheimer's disease (AD) model mice and wild type (WT) mice, which suggests the close correlation between the fecal metabolites and AD pathology and provides new potential biomarkers for the diagnosis of AD.

Describing the fecal metabolome in cryogenically collected samples from healthy participants

“…Several research groups have developed state-of-the-art fecal fingerprinting methods, including chemical labeling with isotopic reagents and high-performance liquid chromatography coupled with high resolution mass spectrometry (HPLC-HRESMS) [18][19][20] , nuclear magnetic resonance (NMR) for compounds like SCFAs and BCAAs [21][22][23] and the use of LC-MS 24,25 and GC-MS 26,27 . In this study, we aim to measure molecules in feces with in-house analytical pipelines using cryogenically drilled samples [28][29][30] , focusing on quality control, sampling procedure and biochemical pathways that can describe feces from healthy volunteers.…”

Current trends in isotope‐coded derivatization liquid chromatographic‐mass spectrometric analyses with special emphasis on their biomedical application

“…Thus, researchers were directed to perform simultaneous ICD for multi‐submetabolomes for the elucidation of the whole metabolomics image of certain biofluids in a certain disease. Recently, Yuan et al () designed a multi‐channel chemical isotope labeling LC–MS strategy for metabolic profiling. The metabolites are categorized based on their reactive functional groups into four submetabolomes: carboxyls, carbonyls, amines, and thiols.…”

Qualitative and Quantitative Analysis of Regional Cerebral Free Fatty Acids in Rats Using the Stable Isotope Labeling Liquid Chromatography–Mass Spectrometry Method

“…FFAs were firstly screened and qualitatively identified in all regions by the CIL-LC-MS method, which has been described in detail in our previous work [28]. The supernatant of 100 mg tissue from each region was equally divided into two parts and the two parts were labeled with DMED or d 4 -DMED, respectively.…”

“…For qualitative analysis, the labeling of DMED and d 4 -DMED was performed by following a previously described method [27,28]. The specific labeling process was as follows: 200 µL ACN was added to brain residues and the mixtures vortexed for 3 min.…”

“…In order to study the maximum possible types of fatty acids, a highly sensitive method is needed. Chemical isotope labeling liquid chromatography-mass spectrometry (CIL-LC-MS) is a suitable technique for this [28]. Utilizing this method, prior to LC-MS analysis, a pair of isotope-coded reagents is used to react with a specific functional group to form derivatives.…”