5‑Diethylamino-naphthalene-1-sulfonyl Chloride (DensCl): A Novel Triplex Isotope Labeling Reagent for Quantitative Metabolome Analysis by Liquid Chromatography Mass Spectrometry

We describe a new set of isotope reagents, ¹²C₄-, ¹²C₂¹³C₂-, and ¹³C₄-5-diethylamino-naphthalene-1-sulfonyl chloride (DensCl), in combination with liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), for improved analysis of the amine- and phenol-containing submetabolome. The synthesis of the reagents is reported, and an optimized derivatization protocol for labeling amines and phenols is described. To demonstrate the utility of the triplex reagents for metabolome profiling of biological samples, urine samples collected daily from a healthy volunteer over a period of 14 days were analyzed. The overall workflow is straightforward, including differential isotope labeling of individual samples and a pooled sample that serves a global internal standard, mixing of the isotope differentially labeled samples and LC-MS analysis for relative metabolome quantification. Comparing to the dansyl chloride (DnsCl) duplex isotope reagents, the new triplex DensCl reagents offer the advantages of improved metabolite detectability due to enhanced sensitivity (i.e., about 1000 peak pairs detected by DensCl labeling vs about 600 peak pairs detected by DnsCl labeling) and analysis speed (i.e., simultaneous analysis of two comparative samples by DensCl vs only one comparative sample analyzed by DnsCl)

5‑Diethylamino-naphthalene-1-sulfonyl Chloride (DensCl): A Novel Triplex Isotope Labeling Reagent for Quantitative Metabolome Analysis by Liquid Chromatography Mass Spectrometry

We describe a new set of isotope reagents, ¹²C₄-, ¹²C₂¹³C₂-, and ¹³C₄-5-diethylamino-naphthalene-1-sulfonyl chloride (DensCl), in combination with liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), for improved analysis of the amine- and phenol-containing submetabolome. The synthesis of the reagents is reported, and an optimized derivatization protocol for labeling amines and phenols is described. To demonstrate the utility of the triplex reagents for metabolome profiling of biological samples, urine samples collected daily from a healthy volunteer over a period of 14 days were analyzed. The overall workflow is straightforward, including differential isotope labeling of individual samples and a pooled sample that serves a global internal standard, mixing of the isotope differentially labeled samples and LC-MS analysis for relative metabolome quantification. Comparing to the dansyl chloride (DnsCl) duplex isotope reagents, the new triplex DensCl reagents offer the advantages of improved metabolite detectability due to enhanced sensitivity (i.e., about 1000 peak pairs detected by DensCl labeling vs about 600 peak pairs detected by DnsCl labeling) and analysis speed (i.e., simultaneous analysis of two comparative samples by DensCl vs only one comparative sample analyzed by DnsCl)

5‑Diethylamino-naphthalene-1-sulfonyl Chloride (DensCl): A Novel Triplex Isotope Labeling Reagent for Quantitative Metabolome Analysis by Liquid Chromatography Mass Spectrometry

We describe a new set of isotope reagents, ¹²C₄-, ¹²C₂¹³C₂-, and ¹³C₄-5-diethylamino-naphthalene-1-sulfonyl chloride (DensCl), in combination with liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), for improved analysis of the amine- and phenol-containing submetabolome. The synthesis of the reagents is reported, and an optimized derivatization protocol for labeling amines and phenols is described. To demonstrate the utility of the triplex reagents for metabolome profiling of biological samples, urine samples collected daily from a healthy volunteer over a period of 14 days were analyzed. The overall workflow is straightforward, including differential isotope labeling of individual samples and a pooled sample that serves a global internal standard, mixing of the isotope differentially labeled samples and LC-MS analysis for relative metabolome quantification. Comparing to the dansyl chloride (DnsCl) duplex isotope reagents, the new triplex DensCl reagents offer the advantages of improved metabolite detectability due to enhanced sensitivity (i.e., about 1000 peak pairs detected by DensCl labeling vs about 600 peak pairs detected by DnsCl labeling) and analysis speed (i.e., simultaneous analysis of two comparative samples by DensCl vs only one comparative sample analyzed by DnsCl)

Development of Isotope Labeling Liquid Chromatography Mass Spectrometry for Mouse Urine Metabolomics: Quantitative Metabolomic Study of Transgenic Mice Related to Alzheimer’s Disease

Because of a limited volume of urine that can be collected from a mouse, it is very difficult to apply the common strategy of using multiple analytical techniques to analyze the metabolites to increase the metabolome coverage for mouse urine metabolomics. We report an enabling method based on differential isotope labeling liquid chromatography mass spectrometry (LC–MS) for relative quantification of over 950 putative metabolites using 20 μL of urine as the starting material. The workflow involves aliquoting 10 μL of an individual urine sample for ¹²C-dansylation labeling that target amines and phenols. Another 10 μL of aliquot was taken from each sample to generate a pooled sample that was subjected to ¹³C-dansylation labeling. The ¹²C-labeled individual sample was mixed with an equal volume of the ¹³C-labeled pooled sample. The mixture was then analyzed by LC–MS to generate information on metabolite concentration differences among different individual samples. The interday repeatability for the LC–MS runs was assessed, and the median relative standard deviation over 4 days was 5.0%. This workflow was then applied to a metabolomic biomarker discovery study using urine samples obtained from the TgCRND8 mouse model of early onset familial Alzheimer’s disease (FAD) throughout the course of their pathological deposition of beta amyloid (Aβ). It was showed that there was a distinct metabolomic separation between the AD prone mice and the wild type (control) group. As early as 15–17 weeks of age (presymptomatic), metabolomic differences were observed between the two groups, and after the age of 25 weeks the metabolomic alterations became more pronounced. The metabolomic changes at different ages corroborated well with the phenotype changes in this transgenic mice model. Several useful candidate biomarkers including methionine, desaminotyrosine, taurine, N1-acetylspermidine, and 5-hydroxyindoleacetic acid were identified. Some of them were found in previous metabolomics studies in human cerebrospinal fluid or blood samples. This work illustrates the utility of this isotope labeling LC–MS method for biomarker discovery using mouse urine metabolomics