Analysis of polar primary metabolites in biological samples using targeted metabolomics and LC-MS

Abstract

Primary metabolites are molecules of essential biochemical reactions that define the biological phenotype. All primary metabolites cannot be measured in a single analysis. In this protocol, we outline the multiplexed and quantitative measurement of 106 metabolites that cover the central part of primary metabolism. The protocol includes several sample preparation techniques and one liquid chromatography-mass spectrometry method. Then, we describe the steps of the bioinformatic data analysis to better understand the metabolic perturbations that may occur in a biological system. For complete details on the use and execution of this protocol, please refer to: Costanza et al.,¹ Blomme et al.,² Blomme et al.,³ Guillon et al.,⁴ Stuani et al.⁵.

Keywords: Mass Spectrometry; Metabolomics.

Graphical abstract

Figure 1

Workflow of the general metabolite sample preparation and analysis

Figure 2

Example of the measurement of isoleucine and leucine

Figure 3

Example of two adjacent peaks that require further confirmation using QC and spike-in to determine which peak corresponds the studied molecule

Figure 4

In rare events, the largest peak may not represent the studied molecule, as shown here for citrulline

Figure 5

Example of data analysis using the MetaboAnalystR package and the MetaboR script The figure shows the metabolite quantification data for HT29 cancer cells after incubation or not with 10 μM metformin. (A) PLS plot outlining control (HT29_Ctrl) and metformin-treated cells (HT29_Met). (B) Volcano plot showing metabolites with at least 2-fold changes in concentration between conditions (HT29_Ctrl and HT29_Met). (C and D) (C) Heatmap and (D) Correlation matrix of all compounds in the dataset.

Figure 6

Absolute quantification for tryptophan in the low μM range. (A) tryptophan calibration curve consisting of quadruplicate measurements of diluted standard. Solid black points indicate values retained for the construction of the curve, while empty circles are excluded values. (B) tryptophan quantification in HT29 cell extracts. Error bars indicate standard deviation of means from N=5 and N=6 biological replicates in HT29 Ctrl and HT29 Met conditions, respectively. The raw data and calibration curves/evaluations for 4-aminobutyric acid and glutamine are in the Supplemental section, Table S6.