Quantification of Isotopologues of Amino Acids by Multiplexed Stable Isotope Resolved Metabolomics using Ultrahigh Resolution Mass Spectrometry Coupled with Direct Infusion

From the Center for Environmental and Systems Biochemistry, University of Kentucky. By Ye Yang, Teresa W-M. Fan, Andrew N. Lane, Richard M. Higashi
Published in Protocols & Methods

Amino acid analysis is an essential component of carbon and nitrogen metabolism in all cells. Conventional methods have insufficient resolution or sensitivity to give reliable quantification and identification in complex mixtures isolated from very small amounts of biological material such as clinical biopsy or low populations of sorted cells from a rodent.  

We are also concerned to determine the synthesis and turnover of the carbon and nitrogen in amino acids, which requires tracers.  We have developed an approach called Stable Isotope Resolved Metabolomics (SIRM) to trace the fate of atoms from a labeled precursor through metabolic transformations.  For amino acid metabolism it is desirable to be able to follow the fate both of the carbon and nitrogen atoms, which normally would requite parallel experiments with 15N and 13C.   However, to cope with small quantities of materials, multiple tracers must be used simultaneously.  

This requires either NMR, which is often not sufficiently sensitive, or mass spectrometry (MS). Many MS platforms cannot resolve a molecule containing 1  13C, 1 15N  or 1 2H atom, which differ from the more common lighter isotope by one neutron. However, adding a neutron to a nucleus does not increase the mass by exactly that of the neutron rest mass, and the mass difference due to the binding energy is isotope specific; ultrahigh resolution MS can resolve these very small isotopic mass differences routinely, and with very high sensitivity.

Makarov-type ("Orbitraps") Fourier transform (FT) instruments are optimized for mid range m/z, and have poorer sensitivity for m/z lower than ca. 100, which includes the small amino acids (e.g. Gly, Ala).  We therefore considered derivatization of amino acids to increase their  m/z values. Several derivatization reagents were considered and compared including N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide, ethyl chloroformate (ECF), 9-Fluorenylmethyl-chloroformate (Fmoc-Cl) and isobutyl chloroformate. Among these,  ECF had the advantages that the reaction occurs at room temperature and in aqueous solution; the reagent and the preparation of the reaction buffer is simple and cost effective; the reaction is fast and the sodiated products show high sensitivity in FTMS analysis. We therefore chose to use the robust ethyl chloroformate derivatization with solvent extraction which removes salt, and makes the resulting mixture compatible with direct infusion FT-MS. Direct infusion also permits higher sensitivity at very high resolution by longer signal averaging than is possible with GC or even UHPLC-MS.  Thus the concept of multiplexed SIRM was brought to fruition, in which more than one isotope to be used in the same experiment.  

To  determine the absolute quantities of each amino acids, in addition to the isotopologue distributions, we used amino acid spiking with labeled amino acids as as internal standards. Since the 15N labeled standards were added before the derivatization, it can be used to correct for variable reaction efficiency, extraction efficiency and even sample degradation.  

Since the ECF reacts with both -NH3 and –COO- groups, this method can also be theoretically used to analyze other important metabolites like polyamines and organic acids.  

The quantitative analysis required systematic optimization of the reaction conditions, determination of the limits of detection and linearity of the response. For most amino acids the responses are linear over a wide dynamic range.

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