This 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.
Bibliographical noteFunding Information:
The authors express their special thanks to Dr. Chi Chen, Associate Professor, and his lab members in the Department of Food Science and Nutrition at the University of Minnesota for their help with UPLC/ESI–QTOF MS, LeeAnn Higgins and Todd W. Markowski from the Center of Mass Spectrometry and Proteomics at the University of Minnesota for their help in sample preparation, analysis, and interpretation of the proteomics data, and Dr. Baraem Ismail and her lab members for helping with size-exclusion chromatography and SDS–PAGE. The authors also thank Dr. Brian Crooker, Professor in the Department of Animal Science at the University of Minnesota for help in obtaining milk from a single cow (numbered 2838) and the Agriculture Experiment Station for their financial support.
© 2020 American Chemical Society.
- Michael addition
- Schiff base
- covalent linkage
- mass spectrometry