Technical Abstract: The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis (2D-GE) on whey samples from a group of 8 cows prior to and 18 hours after infection with Escherichia coli, and to identify differentially expressed milk proteins by peptide sequencing using matrix-assisted laser desorption/ ionization-time of flight (MALDI-TOF) mass spectrometry post source decay (PSD). 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, and complement C3 precursor could be identified in whey samples from healthy cows. Whey samples at 18 hours post infection 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 hours post inoculation were the antimicrobial peptides cathelicidin, indolicidin, and bactenicin 5 and 7, and the proteins ß-fibrinogen, a-2-HS-glycoprotein, S100-A12, and a-1-antiproteinase. Most notable was the detection of the acute phase protein a-1 acid glycoprotein in both healthy and mastitic milk samples. 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 following inoculation with E. coli could provide a rapid and comprehensive survey of milk protein modulation during coliform mastitis and aid in the identification of biomarkers of this disease.