jf9b07978_si_001.pdf (500.9 kB)
Method To Characterize and Monitor Covalent Interactions of Flavor Compounds with β‑Lactoglobulin Using Mass Spectrometry and Proteomics
journal contribution
posted on 2020-02-25, 14:42 authored by Vaidhyanathan Anantharamkrishnan, Gary A. ReinecciusThis
study develops a method to measure the covalent bonds formed
between the side chains and terminal amino acids of β-lactoglobulin
(BLG) and selected flavor molecules (benzaldehyde, citral, or allyl
isothiocyanate) using electrospray ionization mass spectrometry (ESI/MS)
and tandem mass spectrometry (MS/MS). This technique made it possible
to measure increases in molecular weight of BLG as the reaction takes
place (BLG + flavor compound). The observed mass shifts on the reaction
corresponded to either Schiff base or Michael addition reactions between
the chosen flavor compounds and BLG. In the case of citral, sodium
dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed
that these reactions lead to protein cross-linking. A proteomic approach
using MS/MS to identify the sites of post-translational modification
between benzaldehyde and BLG revealed that the lysine groups were
the reaction sites. Interestingly, benzaldehyde was found to react
with several different lysine groups but never more than one of them
per BLG molecule (BLG contains 15 lysine groups/molecule). Furthermore,
adducts with benzaldehyde were not observed at two lysine groups.
Allyl isothiocyanate was found to react with several sites on each
BLG molecule. The ESI/MS methodology in tandem with proteomics yields
a detailed view of flavor/BLG interactions that may offer insights
on minimizing these undesirable reactions in the future.