Pregnancy-Associated Glycoproteins Identification in Skopelos Goat Milk by Means of Mass Spectrometry

The proteins most frequently associated with pregnancy are the pregnancy-associated glycoproteins (PAGs), which are abundantly expressed placental products in species belonging to the order Cetartiodactyla. Multiple PAG isoforms are expressed across different species and stages of gestation, with their expression influenced by factors, such as breed and bodyweight. These proteins can be detected in both blood and milk samples using ELISA or RIA assays, serving as early indicators of pregnancy. The present study aimed to detect PAGs in caprine milk through a non-invasive, high-throughput, mass-spectrometry-based proteomic approach. Milk samples were collected from 20 Skopelos breed goats at 20 and 45 days post-mating. Following appropriate processing for whey protein extraction, the samples underwent tryptic digestion to generate peptides for LC-MS/MS analysis. The proteomic investigation identified five distinct PAGs (caPAG2 on day 20 and caPAG3, caPAG5, caPAG6, and caPAG12,on day 45 post-mating) annotated to Capra hircus, along with 22 additional proteins associated with the fetal–maternal interface, pregnancy progression, and immune-related pathways. These findings demonstrate that LC-MS/MS provides a non-invasive, sensitive, and reliable method for detecting PAGs in caprine milk during the early stages of pregnancy (day 20, as the presence of caPAG2 revealed), applicable to both individual animals and flock-level monitoring.