Normalization of Samples of Limited Amounts in Quantitative Metabolomics Using Liquid Chromatography Fluorescence Detection with Dansyl Labeling of Metabolites

Quantitative
metabolomics requires the analysis of the same or
a very similar amount of samples in order to accurately determine
the concentration differences of individual metabolites in comparative
samples. Ideally, the total amount or concentration of metabolites
in each sample is measured to normalize all the analyzed samples.
In this work, we describe a very sensitive method to measure a subclass
of metabolites as a surrogate quantifier for normalization of samples
with limited amounts. This method starts with low-volume dansyl labeling
of all metabolites containing a primary/secondary amine or phenol
group in a sample to produce a final solution of 21 μL. The
dansyl-labeled metabolites generate fluorescence signals at 520 nm
with photoexcitation at 250 nm. To remove the interference of dansyl
hydroxyl products (Dns-OH) formed from the labeling reagents used,
a fast-gradient liquid chromatography separation is used to elute
Dns-OH using aqueous solution, followed by organic solvent elution
to produce a chromatographic peak of labeled metabolites, giving a
measurement throughput of 6 min per sample. The integrated fluorescence
signals of the peak are found to be related to the injection amount
of the dansyl-labeled metabolites. A calibration curve using mixtures
of dansyl-labeled amino acids is used to determine the total concentration
of labeled metabolites in a sample. This concentration is used for
normalization of samples in the range from 2 to 120 μM in 21
μL with only 1 μL consumed for fluorescence quantification
(i.e., 2–120 pmol). We demonstrate the application of this
sensitive sample normalization method in comparative metabolome analysis
of human cancer cells, MCF-7 cells, treated with and without resveratrol,
using a starting material of as low as 500 cells.