Characterisation of Host Defence Proteins in Milk Using a Proteomic Approach

Besides providing nutrition to the newborn, milk also protects the neonate and the mammary gland
against infection. As well as the six major proteins, bovine milk contains minor proteins, not all of
which have been characterized. In this study, we have subjected bovine skim milk, whey, and milk fat
globule membrane (MFGM) fractions to both direct liquid chromatography-tandem mass spectrometry
(LC-MS/MS), and two-dimensional electrophoresis (2-DE) followed by matrix assisted laser desorption
ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) of individual protein spots to better
characterize the repertoire of minor milk proteins, particularly those involved with host defense. Milk
from peak lactation as well as during the period of colostrum formation and during mastitis were
analyzed to gain a more complete sampling of the milk proteome. In total, 2903 peptides were detected
by LC-MS and 2770 protein spots by 2-DE. From these, 95 distinct gene products were identified,
comprising 53 identified through direct LC-MS/MS and 57 through 2-DE-MS. The latter were derived
from a total of 363 spots analyzed with 181 being successfully identified. At least 15 proteins were
identified that are involved in host defense. These results demonstrate that the proteome of milk is
more complex than has previously been reported and a significant fraction of minor milk proteins are
involved in protection against infection.
Keywords: bovine milk protein • proteomics • 2D electrophoresis • dairy cow