Figure 1—Example of an LC-MS/MS chromatogram obtained by separating a mixture of pure amino acids.
Each amino acid is detected on the basis of a specific signature, called a transition, after separation on an HPLC column. The specificity of the transition makes it possible to analyze co-eluting metabolites (serine and asparagine here) if their mass or fragmentation pattern is different.
An amino acid is a molecule containing carboxyl (COOH; the acid part) and amine (a nitrogen atom linked to at least one carbon atom) functional groups. Among these molecules, about twenty are used in the biosynthesis of peptides and proteins. However, the biological role of amino acids is not limited to protein synthesis. Some can be used as energy sources. Others are neurotransmitters, such as glutamate and glycine. Still others regulate metabolic activity, such as alanine, which inhibits the activity of the pyruvate kinase enzyme in the liver. With their many roles, amino acids sometimes become biomarkers of disease. Plasma concentrations of three amino acids, isoleucine, phenylalanine and tyrosine, for example, are said to be predictive of the development of type 2 diabetes (1).
The CRCHUM amino acid analysis method quantifies 18 of the 20 standard proteinogenic amino acids (all except aspartate and cysteine), as well as citrulline, ornithine, carnitine and the amino acid derivative taurine (Fig. 1).
There are several methods for measuring amino acids. Biochemical methods (colorimetric or fluorometric) were developed to individually quantify the majority of amino acids. Kits also exist to analyze all branched-chain amino acids (leucine, isoleucine, valine) or all of the amino acids without distinguishing
them individually. Valine can also be quantified using an immunoassay (ELISA) kit. However, these methods cannot individually quantify more than one amino acid at a time. To achieve this, nuclear magnetic resonance (NMR) must be used, or the amino acids must be separated using a chromatographic method prior to detection.
Existing separation approaches include high-performance liquid chromatography (HPLC or LC), gas chromatography (GC) and capillary electrophoresis. By chemically modifying amino acids (derivatization), they can be made detectable by absorbance or fluorescence. Separation can thus be coupled with ultraviolet (UV) absorbance detection. HPLC-UV is undoubtedly the most widely used method for measuring amino acids, but other detection methods also exist, including mass spectrometry. This latter detection method, following HPLC separation, was chosen by the CRCHUM metabolomics platform for amino acid determination, i.e., separation by liquid chromatography (LC), coupled with detection by tandem mass spectrometry (MS/MS).
The LC-MS/MS analysis method used at CRCHUM provides absolute values of plasma concentrations (e.g., millimoles per litre). Amino acids are extracted from plasma and analyzed directly, without concentration or chemical modification. Two internal standards are added to samples to reduce the variability introduced by sample preparation and analysis steps. These internal standards are amino acids (d5-glutamine and d5-glutamate) labelled with deuterium, a stable hydrogen isotope. The amino acids are still composed of the same atoms, but their mass is different (+5 -dalton here). The method was developed for plasma but can also be used to quantify the amino acid content of cultured cells or tissue.
Compared with biochemical and immunological methods, the LC-MS/MS approach has the advantage of simultaneously analyzing a large quantity of amino acids, rather than just one or a small group, without distinguishing between the group members. Considering the cost of a kit (> $500/96 points), using LC-MS/MS analysis becomes advantageous after three or more amino acids analyzed (based on 40 samples assayed in duplicate/plate, if the entire plate is used). Compared to the HPLC-UV method, LC-MS/MS analysis is faster (it is not necessary to chemically modify amino acids), more specific and can also be more sensitive. When absorbance is measured, the detector does not distinguish between different molecules; identification is solely based on the retention time observed during chromatographic separation. With tandem mass spectrometry, the identity of amino acids is confirmed by the mass of the amino acid and that of one or more fragments.
Amino acid analysis carried out at CRCHUM is done on 10 µL of plasma or, for cells in culture, on a confluent cell mat of approximately 10 cm2 (1 well of a 6-well plate). Final results are expressed in millimoles per litre (mM) for plasma samples, or in nanomoles per milligram (nmol/mg) of protein for cultured cell samples).
The CRCHUM Metabolomics Core Facility has two LC-MS/MS devices, consisting of a Nexera X2 ultra-high-performance liquid chromatography system (Shimadzu) and a triple quadrupole mass spectrometer (QTrap 6500 and 4000 QTrap; SCIEX).
Any questions?
Bibliography
1) Wang TJ et al. Metabolite profiles and the risk of developing diabetes. Nat Med 2011, 17:448-53
Author: Julien Lamontagne, Core Facilities' Echo committee, with participation by Erik Joly
Featured method: Plasma amino acid LC-MS/MS analysis
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