ABSTRACT: The study reports a differential proteomic analysis of the Mediterranean buffalo milk to evaluate the changes induced by Staphylococcus spp. during a subclinical intramammary infection (IMI). A number of 12 quarter milk samples, 6 of which with somatic cell count (SCC) < 50,000 cells/mL and culture-negative, and the other 6 with SCC ˃ 3,000,000 cells/mL and culture-positive to Staphylococcus aureus (SAU, n=3), SAU, or non-aureus staphylococci (NAS, n=3) was selected. Samples were analyzed using a shotgun proteomics approach, based on filter-aided sample preparation (FASP) followed by LC-MS/MS and label-free analysis. Here, the largest buffalo milk protein dataset described so far was reported. Moreover, the results demonstrated that staphylococcal IMI mostly affected proteins involved in structural functions and in innate immune defense, with changes in their abundance that were generally more intense in SAU than in NAS samples. Further, an increase in the abundance of different cathelicidins was observed, as already reported for other animals with mastitis disease (1,2). (1) Addis MF, Pisanu S, Marogna G, Cubeddu T, Pagnozzi D, Cacciotto C, et al. Production and release of antimicrobial and immune defense proteins by mammary epithelial cells following Streptococcus uberis infection of sheep. Infect Immun. 2013;81: 3182–3197. (2) Addis MF, Tedde V, Dore S, Pisanu S, Puggioni GMG, Roggio AM, et al. Evaluation of milk cathelicidin for detection of dairy sheep mastitis. J Dairy Sci. Elsevier; 2016;99: 6446–6456. In conclusion, our results provide the first in depth characterization of buffalo milk proteins, describe the changes induced by SAU and NAS subclinical intramammary infection and suggest indications to reveal subclinical staphylococcal mastitis in buffalo by the milk proteome investigation.