Disulfide Bonds detection and characterization in milk proteins by 1-DE Electrophoresis and Mass Spectrometry approaches

Various protein cross-linking reactions leading to molecular polymerization and covalent aggregates have been described in processed foods. They are an undesired side effect of processes designed to reduce bacterial load, extend shelf life, and modify technological properties, as well as being an expected result of treatments designed to modify raw material texture and function. Although the formation of these products is known to affect the sensory and technological properties of foods, the corresponding cross-linking reactions and resulting protein polymers are commonly overlooked in mass spectrometry (MS)-based proteomics approaches [1]. Description of these structural modifications is crucial for evaluating how their generation can be related to food processing conditions and quality parameters. In the current study, we have combined 1-DE electrophoresis with nanoliquid chromatography coupled with electrospray quadrupole-Orbitrap tandem mass spectrometry (nLC-ESI-Q-Orbitrap-MS/MS) to characterize protein disulfide bonds in different commercial milk products as well as in raw milk subjected to heating for various times. By using dedicated bioinformatic software, both inter- and intra-disulfide-linked peptides were identified and structurally characterized in abundant protein components of milk.