Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by¹³C-/¹²C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Abstract

Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by 13 C-dansyl and 12 C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner.

Relative quantification of amine-containing metabolites using isobaric N,N-dimethyl leucine (DiLeu) reagents via LC-ESI-MS/MS and CE-ESI-MS/MS

“…25, 26 Stable isotope labeling can be categorized into two groups, mass-difference labeling and isobaric labeling. Mass-difference labeling, such as 12 C-/ 13 C-methyl acetimidate, 27 dansylation, 28, 29 and formaldehyde dimethylation, 30 introduces a fixed mass difference for the same metabolite in a MS spectrum. Relative quantification is achieved by comparing extracted ion chromatogram peak areas of the heavy and light isotopic forms of the same metabolite.…”

Development of versatile isotopic labeling reagents for profiling the amine submetabolome by liquid chromatography–mass spectrometry

“…For example, Guo and Li developed a CIL method based on 12 C 2 -and 13 C 2 -dansyl chloride (DnsCl) [1]. Although the method allows for rapid and accurate quantification of amine-containing metabolites [26][27][28][29], metabolite identification, particularly for unknown metabolites, is a challenge due to the lack of structural information in the MS/MS spectra of dansyl labeled metabolites. Tsukamoto et al developed H 6 -/D 6 -7-(N,Ndimethylaminosulfonyl)-4-(aminoethyl)-piperazino-2,1,3-benzoxadiazole (H 6 -/D 6 -DBD-PZ-NH 2 ) to profile fatty acids in rat plasma samples [8] and Shimbo et al reported the use of p-N,N, N-trimethylammonioanilyl N 0 -hydroxysuccinimidyl carbamate iodide (TAHS) and D 3 -TAHS for amino acid quantification [30].…”

Biomarker Discovery and Translation in Metabolomics

“…A variety of internal standardization methods using isotope labeled compounds ( 2 H, 13 C, 15 N) as well as external standardization methods are also available to provide much better reproducibility. First developed to provide improved quantitation in the field of proteomics, the use of mixtures of metabolites containing light and heavy isotopes is a popular method for relative and absolute quantitation of metabolites using MS methods [47–50]. Targeted methods that use such internal standards compensate ion-suppression effects and provide reliable metabolite quantitation.…”