Quantitation of human milk proteins and their glycoforms using multiple reaction monitoring (MRM)

Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. Graphical Abstract The glycopeptides EICs generated from QQQ.