PMC

Resolution comparison between GC-Orbitrap/MS and GC-TOF/MS (triple-silylated glutamic acid). Higher resolution helps to reduce possible formulae based on the molecular weight. After considering the number of carbons in the metabolite and silylation groups, the number of carbons was set to 14–15, and the number of silicon atoms was set to 2–3, allowing for possible hydrogen, oxygen, nitrogen and sulfur. The metabolite was identified as triple-silylated glutamic acid with molecular formula as C₁₄H₃₄NO₄Si₃. The accurate mass is 346.1796. The mass derivation for GC-TOF/MS (upper) was 18.4 ppm and ranked as the seventh candidate, while the mass derivation was for GC-Orbitrap/MS (lower) was −0.8 ppm, and ranked as the first of all possible candidates.

Extracted ion chromatogram (EIC) of m/z 131.08 and mass spectrum of silylated yeast extracts, 2,3-dihydroxyisovalerate, and their combination. Bottom panels (in red color) are the EIC and the mass spectrum from yeast extracts, the middle panels (in green) are the EIC and the mass spectrum from the pure compound (2,3-dihydroxyisovalerate) after derivatization, and top panels (in black) are the EIC and the mass spectrum from yeast extract spiked with pure compound (2,3-dihydroxyisovalerate). The parent molecule has five carbons, and the fragments m/z of 131.0887 and 292.1346 have three carbons and two carbons, respectively, in addition to the silylation group carbons (top right panel). The IROA fragment peak pairs, 131/134 and 292/294, are clearly seen, but their ratio is different between the bottom and top panels due to spiking with the unlabeled 2,3-dihydroxyisovalerate.

Comparison among three different CI reagent gases. Three reagent gases (pure ammonia (top in red color), 10% ammonia in methane (middle in purple color), and pure methane (bottom in black color)) in positive CI were compared for analysis with unlabeled S. cerevisiae extract samples. The left panels are the extracted ion chromatograms (EIC) for the [MH]⁺ ion of triple-silylated glutamine with m/z of 363.195. 10% ammonia in methane showed highest intensity (Int) of the EICs. The right panels are mass spectra of glutamine under three different reagent gases. Pure ammonia showed highest intensity artifact m/z peak of 90.0735, pure methane showed highest intensity of MH-CH₄ fragment ion (347.1638), while 10% ammonia in methane showed highest intensity of [MH]⁺ ion of triple-silylated glutamine (363.1949).

Expanded IROA metabolite identification procedure with in silico fragmentation. Isoleucine is used as an example here. Positive CI-IROA distinguishes unknown metabolites of biological origin from any artificial peaks, and identifies the molecular ion, number of carbons (n) in the metabolite from the dalton difference of the M₀ and M_0+n IROA peaks, and number of silylation groups attached to the metabolites. The exact mass of M₀ molecular ion is combined with the EI mass spectrum found from the 95% ¹²C IROA sample as inputs into CFM-ID to search for potential IDs. The structural identity and its molecular ion can be validated/confirmed from the CI-IROA molecular ion, and the CFM-ID in silico EI fragmentation pattern can be confirmed from the IROA EI pattern.