Key changes in bovine milk immunoglobulin G during lactation: NeuAc sialylation is a hallmark of colostrum immunoglobulin G N-glycosylation Key changes in bovine milk immunoglobulin G during lactation: NeuAc sialylation is a hallmark of colostrum immunoglobulin G N-glycosylation

We monitored longitudinal changes in bovine milk IgG in samples from four cows at 9 time points in between 0.5-28 days following calving. We used peptide-centric LC-MS/MS on proteolytic digests of whole bovine milk, resulting in the combined identification of 212 individual bovine milk protein sequences, with IgG making up >50% of the protein content of every 0.5 d colostrum sample, which reduced to ≤3% in mature milk. In parallel, we analysed IgG captured from the bovine milk samples to characterise its N-glycosylation, using dedicated methods for bottom-up glycoproteomics employing product ion-triggered hybrid fragmentation. The bovine milk IgG N-glycosylation profile was revealed to be very heterogeneous, consisting of >40 glycoforms. Furthermore, these N-glycosylation profiles changed substantially over the period of lactation, but consistently across the four individual cows. We identified NeuAc sialylation as the key abundant characteristic of bovine colostrum IgG, significantly decreasing in the first days of lactation, and barely detectable in mature bovine milk IgG. We also report, for the first time to our knowledge, the identification of subtype IgG3 in bovine milk, alongside the better-documented IgG1 and IgG2. The detailed molecular characteristics we describe of the bovine milk IgG, and their dynamic changes during lactation, are important not only for the fundamental understanding of the calf’s immune development, but also for understanding bovine milk and its bioactive components in the context of human nutrition.