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In situ generation of milk protein-derived peptides in drying

Ho, Chin H., Chang, Chai J., Liu, Wen B., Peh, Huo C., Chen, Shuen E., Chen, Hui Y., Ho, Tsung H., Chen, Ming T., Nagahata, Hajime
Journal of dairy research 2010 v.77 no.4 pp. 487-497
dairy cows, secretion, beta-casein, peptides, lactoglobulins, lactoferrin, bovine serum albumin, mammary glands, protein content
Our previous studies demonstrated prompt elevation of proteinase activity in mammary secretion of drying-off cows and goats. The current study examined the progressive changes in composition of cow mammary secretion following drying-off and, in parallel, characterized the mode of peptide neogenesis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-electrospray-ionization (LC-ESI) MS/MS. The results show that the percentage of casein of total milk protein at time of drying-off was 76%, which dropped to 41%, 24%, and 16%, respectively, 1, 2, and 3 weeks after drying-off. Levels of β-lactoglobulin and α-lactoalbumin in mammary secretions of drying-off cows decreased prominently while levels of lactoferrin, BSA, and casein derived-proteins increased concomitantly compared with regular milk. A fractionation procedure was applied to remove molecules larger than 10 kDa before MALDI-TOF MS and LC-ESI MS/MS and the results show that the MALDI-TOF MS peptide profile of mammary secretion ranging from m/z 600 to 4000 was apparently modified after drying-off for 1 week, whereas species 1590 m/z and 2460 m/z were most obviously enriched compared with regular milk. LC-ESI MS/MS results were used to map peptide sequence with Mascot search server and under no post translational modification to reduce database size and 202 novel β-casein-derived peptides were successfully identified in mammary secretion after drying-off for 1 week in contrast to regular milk. Accordingly at least 48 additional cleavage positions were assigned on β-casein for mammary secretion. Among the 202 novel peptides, 5 are homologous with confirmed opioid agonists, angiotensin 1-converting enzyme inhibitors, or immuno-modulators. In conclusion, peptides are released in situ from milk proteins within short intervals following drying-off in cows. They might play roles in the transition of mammary glands from lactating to non-lactating. With specified post-translational modifications and focused functional screening, novel peptides are yet to be discovered in dry cow mammary secretion.