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Supplementary Material
1.
RATIONALE FOR AMINO ACIDS TARGETED
The five amino acids investigated in this study (proline, phenylalanine, leucine, valine, and isoleucine)
were selected because their hydrogen
isotope compositions are among the most
reliable to interpret based
on preparatory and analytical considerations. These amino acids do not
degrade during hydrolysis, even in
the presence of oxygen (Table S4; Silverman et al., 2022), nor do they experience hydrogen exchange with
aqueous medium
during hydrolysis or derivatization (Figs. S
9
and S
1
0
). Moreover, they exhibit consistent
baseline chromatographic
separation, and their high
abundances in cells and great ionization efficiencies
result in relatively intense chromatographic peaks that can be
measured with reasonable precision (Fig.
S1; Table S2).
Of the remaining 15 common biological amino acids, some remain isotopically faithful through
preparation, while others may not be possible
to target for δ
2
H analysis. A thorough review of the
preparatory and analytical considerations for isotopic analysis of each amino acid is
provided in Silverman
et al. (2022) and briefly summarized here in the context of our methodological approach. Lysine is stable
during
hydrolysis and derivatizes sufficiently to an MOC methyl ester, thus is a promising target for
futu
re δ
2
H analyses. Alanine and glycine are
also stable during hydrolysis but co
-
elute on a ZB
-
5ms
column when derivatized as MOC methyl esters; however, these amino acids can be
separated when using
different derivatization reactions and/or column stationary phases (e.g., Corr et al., 2007; Walsh et al., 2014).
Asparagine and glutamine quantitatively deamidate to aspartic acid and glutamic acid during hydrolysis
(Wright, 1991), while the acidic
amino acids and tyrosine experience moderate to significant hydro
gen
exchange with aqueous medium (Fig. S
9
; Hill and Leach, 1964), so the
δ
2
H values of these amino acids
should be interpreted with caution. The peak shape of threonine was often
problematic in our analyses
(Fig.
S1), potentially related to incomplete derivatization of
threonine’s
hydroxyl group and
its consequent
interaction with the
column stationary
phase (H
ek, 1991). Histidine and serine have relatively low reaction yields when
derivatized to MOC esters (Walsh et al., 2014), but the
former amino acid is sometimes still accessible for
analysis. Arginine is difficult to derivatize with the majority of common GC
-
based
derivatization reactions
(Silverman et al., 2022). Cysteine an
d methionine experience extensive oxic degradation during hydrolysis
(Phillips et
al., 2021), although the mechanism of loss suggests that their hydrogen isotope ratios may not
be affected (Silverman et al., 2022), which was
corroborated for methionine by hydrolysis tests in this
study (Table S4). Tryptophan also degrades extensively during hydrolysis and is
difficult to recover for
most applications.