In situ generation of milk protein-derived peptides in drying-off cows.
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.
Ho
Chin H
CH
Department of Animal Science, National Chung Hsing University, Taichung 402, Taiwan ROC.
Chang
Chai J
CJ
Liu
Wen B
WB
Peh
Huo C
HC
Chen
Shuen E
SE
Chen
Hui Y
HY
Ho
Tsung H
TH
Chen
Ming T
MT
Nagahata
Hajime
H
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle / Q000502:physiology*; D005260:Female; D005374:Filtration; D007774:Lactation / Q000502:physiology*; D008321:Mammary Glands, Animal / Q000502:physiology; D008892:Milk / Q000737:chemistry* / Q000502:physiology; D008894:Milk Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008894:Milk Proteins; D010455:Peptides