Defining the Porcine Colostral Proteome: Changes in the Array of Proteins from Colostrum to Mature Milk

Abstract

The importance of colostrum (first milk) for immunological and nutritional support of newborn mammals is well established. Many bioactive peptides are present in milk at higher concentrations than in maternal circulation. Transmission of such factors from mother to offspring as a specific consequence of nursing is characterized as lactocrine communication. Because milk-borne, lactocrine-acting factors affect patterns of gene expression in neonatal somatic tissues, including the reproductive tract and heart, it is important to understand the biochemical nature of colostrum/milk. Data for relaxin, a prototypical lactocrine-acting factor in porcine colostrum, indicate that transmission of such factors is significant prior to gut closure in the neonatal pig. Additionally, amino acid sequences encoding potentially bioactive peptides are encrypted within porcine milk proteins, raising the possibility that proteolytic cleavage in the gut could release such factors into circulation after consumption. The array of proteins/peptides constituting the porcine colostral proteome has not been defined. Objectives of this study were to employ two-dimensional gel electrophoresis (2DE) and image analysis to: (1) define the porcine colostral proteome on lactation day (LD) 0; and (2) determine if and how this proteome changed from LD 0 to LD 6. Colostrum (LD 0) and milk (LD 6) samples were obtained from six lactating sows. Protein was extracted from individual samples and total protein concentrations were determined. Extracted proteins were prepared for and subjected to both standard SDS-PAGE (10% total monomer) and 2DE. For 2DE, first dimension separations were carried out using pH 3-10 immobilized pH gradient strips followed by SDS-PAGE using gradient polyacrylamide gels (10-20% total monomer). Individual samples were run on duplicate 2DE gels and stained with Sypro RUBY. Digital images of individual gels were obtained and analyzed using a Typhoon 9400 digital scanner and PDQuest 2-D Analysis Software. Total protein concentrations for colostrum (LD 0) and milk (LD 6) were 8.5 mg/ml and 8.3 mg/ml. Standard SDS-PAGE analyses revealed distinct differences in the distribution of protein bands between LD 0 and LD 6. PDQuest analyses identified consistent qualitative and quantitative differences between colostrum from LD 0 and milk from LD 6. Systematic analyses of the primary amino acid sequences of porcine milk proteins using the BIOPEP program and related database (www.uwm.edu.pl/biochemia/index_en.php) revealed that potentially bioactive peptides are encrypted within porcine milk proteins. Thus, newborn pigs that nurse obtain a complex mixture of proteins and peptides from birth that changes with time during a period of neonatal life recognized to be critical for female reproductive tract development. Gilts deprived of colostrum for two days from birth exhibit altered gene expression patterns essential for normal development of female reproductive tract tissues. The presence of encrypted peptides in porcine colostrum increases the complexity of the porcine milk proteome. These studies provide a framework for future efforts to be aimed at identification of colostral proteins and peptides that affect lactocrine programming of neonatal development.