Identification of Bovine Pregnancy-Specific Milk Proteins Using Proteomics.

Abstract

Early pregnancy diagnosis is an essential component of reproductive plan in dairy cattle, because the long term of non-pregnancy results in longer dry period and low milk productivity. This study was conducted to identify early pregnancy-specific milk proteins in bovine by comparing milk samples collected from cattle during pregnancy (Day 30 and 50) and non-pregnancy. In this experiment, global proteomics approach was exploited by using 2-D gel electrophoresis and mass spectrometry and milk from pregnant and non-pregnant Holstein dairy cattle was analyzed to sort out the pregnancy-specific milk proteins. Milk samples were centrifuged following lowering to pH 4.6 to remove casein proteins just after collection and frozen until use. The first dimension, pH 3.0-10.0 strip was used and 2 mg of milk protein sample was loaded onto a cup at the anodic side. Automatic isoelectric focusing was carried out by 1.5 ¡¿ 105 Vh. Voltage started from 100 V and gradually increased to a final voltage of 8000 V. The second dimensional separation was performed on 8-16% linear gradient SDS polyacrylamide gels, and the gels were stained with colloidal Coomassie brilliant blue. The stained gels were scanned and the images were analyzed to detect variations in protein spots between non-pregnant and pregnant milk protein spots using an Image Master followed by MALDI TOF-MS. Analysis of the 2-DE gel image exhibited a total of approximately 600~700 protein spots. Among them, 16 protein spots turn out to be differentially expressed in the comparison of pregnant bovine milk samples with non-pregnant milk samples, of which 14 spots were pregnancy-specific and 2 spots down-regulated in the pregnant milk sample. Pregnancy-specific proteins were identified as serum albumin precursor, IgG1 heavy chain constant region, conglutinine precursor, epithelial keratin 10 and kelch-like ECH-associated protein. Though some identified spots were abundant milk or serum proteins, their molecular weights and pI values were different from main milk or serum proteins suggesting that these proteins may be pregnancy-specific subunits or fragments of albumin and IgG. Our results revealed composite profiles of several milk proteins related to bovine early pregnancy by 2-D PAGE analysis and implied the possible use of these milk proteins for the detection of bovine early pregnancy.