During food processing insult-induced crosslinks form between proteins, which profoundly influence the nutritional value and properties of food. One crosslink that forms with heating and basic pH is lysinoalanine. Despite the adverse effects of lysinoalanine, its formation and biological fate are not well understood. Directly mapping insult-induced protein-protein crosslinks has many challenges.
Using mass spectrometry, we aim to identify a diagnostic fragmentation pattern of lysinoalanine to help to map its location in food-related samples.
Simple peptide models of a lysinoalanine, disulfide and lanthionine crosslinks have been isolated and characterised from MALDI-TOF/TOF fragmentation spectra. The model protein β-lactoglobulin was heated at pH 12 to form crosslinks and analysed using LC-QTOF-MS/MS. The locations of crosslinks in the protein can be identified by comparing acquired data to the theoretical masses of crosslinked peptides.
The crosslink models suggest the lysinoalanine preferentially fragments at the α-carbon and β-carbon. Trends in the model crosslinks show increasing the length of heating and pH both results in more lysinoalanine and lanthionine crosslinks forming and fewer disulfide bonds form. Increasing these conditions also results in lysinoalanine forming in favour of lanthionine. Identifying crosslinks in the milk protein, β-lactoglobulin, formed as a result of from thermal and alkaline treatment is been undertaken through LC-QTOF-MS. Characterisation of crosslinks in the protein will help validate the fragmentation pattern and trends identified in the peptide crosslink models.
We have identified a putative fragmentation pattern to help map the location of crosslinks within a milk protein. Knowing the location of the crosslinks gives the opportunity for these structural features to be included in digestion models. Varying reaction conditions has enabled us to investigate a proteins’ susceptibility to from crosslinks. This guides us on how altering food preparation procedures can influences lysinoalanine formation and the opportunity to increase foods’ nutritional value.