Proteomic Analysis of Differentially Expressed Proteins in Bovine Milk During Experimentally Induced Escherichia coli Mastitis

Abstract

The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis on whey samples from a group of 8 cows before and 18 h after infection with Escherichia coli and to identify differentially expressed milk proteins by peptide sequencing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry post source decay. Only proteins present in whey fractions of all 8 cows were sequenced to avoid reporting a protein response unique to only a subset of infected cows. Despite the overwhelming presence of casein and beta-lactoglobulin, the low abundance proteins transthyretin, lactadherin, beta-2-microglobulin precursor, alpha-1-acid glycoprotein, and complement C3 precursor could be identified in whey samples from healthy cows. Whey samples at 18 h postinfection were characterized by an abundance of serum albumin, in spots of varying mass and isoelectric point, as well as increased transthyretin and complement C3 precursor levels. Also detected at 18 h postinoculation were the antimicrobial peptides cathelicidin, indolicidin, and bactenecin 5 and 7, and the proteins beta-fibrinogen, alpha-2-HS-glycoprotein, S100-A12, and alpha-1-antiproteinase. Most notable was the detection of the acute phase protein alpha-1-acid glycoprotein in mastitic whey samples, a result not previously reported. In contrast to methods used in previous proteomic analyses of bovine milk, the methods used in the current study enabled the rapid identification of milk proteins with minimal sample preparation. Use of a larger sample size than previous analyses also allowed for more robust protein identification. Results indicate that examination of the protein profile of whey samples from cows after inoculation with E. coli could provide a rapid survey of milk protein modulation during coliform mastitis and aid in the identification of biomarkers of this disease.

A peptidomic approach to biomarker discovery for bovine mastitis

“…Clinical mastitis causes decreases in caseins, α-lactalbumin and β-lactoglobulin [10] and increases in serum albumin and serotransferrin. Further studies have identified additional proteins with altered expression in milk from dairy cows with experimental induction of mastitis [11][12][13]. These studies have revealed increased concentrations of chaperonins, DNA-binding proteins, enzymes, transport proteins and structural proteins, complement factors C3 and C4, lactoferrin, transferrin, apolipoprotein AI, fibrinogen, glycosylation-dependent cell adhesion molecule-1, peptidoglycan recognition receptor protein and cyclic dodecapeptide-1.…”

Effects of the processing methods of corn grain and soybean meal on milk protein expression profiles in dairy cows

“…Further, phosphorylation and glycosylation of bovine κ-CN have also been characterized by Holland et al (2006). However, the published comparative proteomic analyses have mostly focused on host defense proteins in bovine milk that protect the neonate/mammary gland against infection or diagnostic markers of mastitis in affected animals (Boehmer et al, 2008). Few published reports have studied the effects of dietary nutrient supply on expression profile changes in milk protein composition using proteomics.…”

“…Milk production is a defining feature of dairy cows. Several factors, such as cow health, environment, lactation stage, genetics and diet, can influence milk protein content and yield (Schingoethe, 1996;Boehmer et al, 2008). In addition, synchronizing the degradation of ruminal carbohydrate and protein by changing diet ingredients can theoretically maximize microbial protein synthesis to support milk production (Casper et al, 1999;Cole and Todd, 2008).…”

Effects of duodenal infusion of free α-linolenic acid on the plasma and milk proteome of lactating dairy cows

“…Albumin is a plasma protein whose elevation in milk indicates disintegration of the blood-milk barrier (Vangroenweghe et al, 2004). The observed elevation in milk albumin after high-dose post-ruminal infusion of LNA was consistent with changes in the protein composition of milk seen during naturally and experimentally induced mastitis (Hogarth et al, 2004;Boehmer et al, 2008;Boehmer et al, 2010;Ibeagha-Awemu et al, 2010). In the current study, the elevated albumin and SCC in milk were accompanied by disintegration of the blood-milk barrier, suggesting a metabolic stress of mammary gland owing to the LNA infusion.…”