Advantages of exact mass analysis
Nominal mass GC-MS analyses rely on two main criteria: retention index, and mass spectrum (Garcia & Barbas, 2011). A well-recognised difficulty with mass spectrum analysis and comparisons in biological samples is co-elution of analytes, leading to mixed mass spectra. To address this problem, deconvolution algorithms can be used (e.g. in ChromaTOF®, or the deconvolution plugin in TraceFinder®) (Du & Zeisel, 2013; H. Lu, Liang, Dunn, Shen, & Kell, 2008) or alternatively, data extraction can focus on individual target m/z, e.g., in MetabolomeExpress (Carroll, Badger, & Harvey Millar, 2010). However, there remains a risk that co-elution leads to erroneous identification and quantitation when common m/z nominal mass features are present. Here, we have covered this aspect by searching systematically co-eluting nominal masses m/z (Table 1, Fig. 3). Rather common plant metabolites are concerned by this problem (e.g., malate, serine, etc.). Exact mass offers an efficient way to avoid it since exact mass values of targeted m/z features are clearly different. In addition, the isotopic pattern reflects elemental composition (Fig. 2) and can be used as a post-hoc verification. Erroneous identification is therefore much less probable. Here, using Arabidopsis samples, there was only one case of misidentification: iodo-tyrosine 3TMS has been identified by TraceFinder® at 16.12 min (Fig. 6c). This molecule is very unlikely since enzymes of iodo-tyrosine metabolism (thyroperoxidase and iodo-tyrosine deiodinase) are absent in plants (Phatarphekar, Buss, & Rokita, 2014; Taurog, 1999). Our database available in Supplementary material has been curated accordingly. Coincidentally, several unusual sugar-containing molecules with similar retention time can generate fragments (m/z features) with the same exact mass. For example, rutinose 6TMS 1Meox elutes just before sucrose 8TMS (16.17 min here) and can form a target fragment at 218.1033 like iodo-tyrosine (Fig. SX). The observed signal is very low (10-6 that of sucrose 8TMS), suggesting it is a minor compound. Further work is needed (for example using MS2 on a purified fraction where this compound is more concentrated and thus generates well-visible MS² fragments) to determine what compound generates a signal similar to iodo-tyrosine in Arabidopsis samples.
Another advantage of exact mass analyses is the resolution of isotopologues. This is of particular interest to identify13C-molecules upon double-labelling (typically13C-15N, or13C-33S) since the signal associated with 13C mass excess (+1.003355 Da) is readily visible. This allows direct quantitation of 13C isotopologues unlike in nominal mass analyses where all m +1 isotopologues are under the same peak. In a recent study, the systematic, non-targeted analysis of 13C isotopologues (+1.003355 Da, +2.006710 Da, +3.010065 Da, etc.) has been used with high resolution LC-MS to look at variations in leaf metabolites with CO2 and O2 gaseous conditions (Abadie, Lalande, Limami, & Tcherkez, 2021). For34S-isotopologues, there is an interference with30Si (silicium being carried by trimethylsilyl groups). Despite this limitation, it is worth noting that whenever mass resolution is sufficiently high (in particular for low m/z values), it is possible to identify S-containing fragments (Fig. 5). Resolution is less of an issue upon labelling since 34S would prevail over 30Si and thus the peak apex would be closer to the mass excess of 34S (+1.995796 Da).