Gas chromatography coupled to mass spectrometry (GC/MS) is emerging as a powerful tool for profiling large numbers of primary metabolites, 12,34,39,40 and we are incorporating this approach into our program. The favorable attributes of GC/MS include high reproducibility (low analytical variance), standardized technique, and high separation efficiencies. High separation efficiencies allow for the separation of complex mixtures, and are achieved with long (30 to 60 m) capillary columns (internal diameters 75 to 320 ?m). GC/MS is commonly used in conjunction with electron ionization and requires the analyte to be volatile, thermally stable, and energetically stable. Many important biological analytes are polar and nonvolatile; therefore, they must be first chemically modified or derivatized prior to GC/MS analysis.  

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Figure 3 GC/MS metabolic profiles of a polar M. truncatula root extract that illustrates the elution regions of various metabolite classes. The (a) normalized chromatogram is dominated by several peaks, but the (b) expanded view of the same root profile reveals a substantially large amount of information not apparent at first glance.

We are using GC/MS for profiling primary metabolites in M. truncatula. This approach allows for the simultaneous profiling of approximately 300 to 500 components, including amino acids, organic acids, monosaccarides, disaccarides, alcohols, and aromatic amines. 12,31 A typical GC/MS profile of a M. truncatula root extract is shown (Fig. 3). The figure illustrates the naturally occurring relative abundances of metabolites visualized by GC/MS while also providing an expanded view of the profiles revealing the large amount of information contained within the data.

A large number of primary metabolites can be readily identified because most of these compounds are commercially available. Standard compounds are derivatized, co-chromatographed, and the data are deposited into databases. Unknown metabolites are identified by matching chromatographic retention times and mass spectra to that of known compounds in the databases. 41,42 Mass spectral identification is performed by matching target spectra with commercial libraries such as The National Institute of Standards and Technology (NIST) library or custom libraries constructed in-house using authentic standards. There are several computer algorithms that automate the process of database searching and identification. 43-45 An example includes the Automated Mass Spectral Deconvolution and Identification Software (AMDIS) provided with many Hewlett Packard GC/MS instruments. 46 We exploit both custom and commercial libraries for metabolite identifications. Using this approach we have identified a large number (~130 currently) of primary metabolites in M. truncatula (Fig. 4). This method has also been used to compare the profiles of various M. truncatula tissues.

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Figure 4 GC/MS metabolic profile of a polar M. truncatula root extract that provides the identification for many of the root components. Individual components are identified by matching their mass spectra to those in databases or by comparison with authentic samples. Using this approach we have identified a large number (>130 currently) of primary metabolites in M. truncatula.

The primary limitation associated with GC/MS is the need for derivatization. Derivatization introduces additional complexity to the system and is not 100% efficient. Inefficient reactions result in the presence of multiple derivatized forms of the same compound. For example, we can detect three different derivatization products of the amino acid asparagine (mw = 132) in M. truncatula roots (Fig. 4). These include asparagine, N,O-TMS (mw = 276), asparagine, N,N,O-TMS (mw = 348), and asparagine, N,N,N,O-TMS (mw = 420). Inefficiency of the derivation reactions also limits the lower concentration range of analytes that can be profiled. Finally, derivartization is not capable of achieving volatility for all compounds, such as many of the flavonoid glycosides. If derivatization is successful and the analyte is volatilized, it must still remain energetically stable enough to be detected. If the compound is not stable, it will fragment and molecular weight information may be lost, thereby complicating identification.

References