A novel proteomic approach for the identification and relative quantification of disulfide-bridges in the human milk proteome

This study explores the disulfide bridges present in the human milk proteome by a novel approach permitting both positional identification and relative quantification of the disulfide bridges. Human milk from six donors was subjected to trypsin digestion without reduction. The digested human milk proteins were analyzed by nanoLC-timsTOF Pro combined with data analysis using xiSEARCH. A total of 85 unique disulfide bridges were identified in 25 different human milk proteins. The total relative abundance of disulfide bridge -containing peptides constituted approximately 5% of the total amount of tryptic-peptides. Seven inter -molecular disulfide bridges were identified between either alpha-lactalbumin and lactotransferrin (5) or alpha S1-casein and kappa-casein (2). All cysteines involved in the observed disulfide bridges of alpha-lactalbumin and lactotransferrin were mapped onto protein models using AlphaFold2 Multimer to estimate the length of the observed disulfide bridges. The lengths of the disulfide bridges of lactotransferrin indicate a potential for multi- or poly-merization of lactotransferrin. The high number of intramolecular lactotransferrin disulfide bridges identified, suggests that these are more heterogeneous than previously presumed. Significance: Disulfide -bridges in the human milk proteome are an often overseen post-transaltional modification. Thus, mapping the disulfide -bridges, their positions and relative abundance, are valuable new knowledge needed for an improved understanding of human milk protein behaviour. Although glycosylation and phosphorylation have been described, even less information is available on the disulfide bridges and the disulfide -bridge derived protein complexes. This is important for future work in precision fermentation for recombinant production of human milk proteins, as this will highlight which disulfide -bridges are naturally occouring in human milk proteins. Further, this knowledge would be of value for the infant formula industry as it provides more information on how to humanize bovine -milk based infant formula. The novel method developed here can be broadly applied in other biological systems as the disulfid-brigdes are important for the structure and functionality of proteins.