Targeted Proteomics Confirms Bovine Milk Peptide Signatures for Differentiating Healthy from Subclinical Mastitic Milk Infected with <i>Streptococcus uberis</i> and <i>Staphylococcus aureus</i>

Bovine subclinical mastitis (SCM), an asymptomatic mammary inflammation, can lead to milk spoilage, premature culling of animals, and compromised milk quality. Our study aimed to identify and establish the functionality of milk proteins as potential markers for SCM state utilizing multiple reaction monitoring mass spectrometry (MRM-MS). In our study, 464 protein groups were confidently identified, of which 215 protein groups showed relatively different expression between healthy (H) and SCM. The MRM assay was used to quantify peptides NLQI and SLFS (lactophorin), EITQ (serum-amyloid-A), WEEA (leukotriene-A4-hydrolase), and LISD (kininogen) on a cohort of 30 animals in each animal group for evaluating their diagnostic potential and discriminating performances. Compared to one protein's effectiveness as an individualistic marker, the synergistic pattern of multiple peptides achieved better discriminating power toward SCM from healthy individuals with sensitivity (>0.90) and specificity (>0.90). The study's results established peptide signatures in milk, distinguishing the SCM from the H group.