DIA mass spectrometry characterizes urinary proteomics in neonatal and adult donkeys

Health monitoring is critical for newborn animals due to their vulnerability to diseases. Urine can be not only a useful and non-invasive tool (free-catch samples) to reflect the physiological status of animals but also to help monitor the progression of diseases. Proteomics involves the study of the whole complement of proteins and peptides, including structure, quantities, functions, variations and interactions. In this study, urinary proteomics of neonatal donkeys were characterized and compared to the profiles of adult donkeys to provide a reference database for healthy neonatal donkeys. The urine samples were collected from male neonatal donkeys on their sixth to tenth days of life (group N) and male adult donkeys aging 4-6 years old (group A). Library-free data-independent acquisition (direct DIA) mass spectrometry-based proteomics were applied to analyze the urinary protein profiles. Total 2179 urinary proteins were identified, and 411 proteins were differentially expressed (P < 0.05) between the two groups. 104 proteins were exclusively expressed in group N including alpha fetoprotein (AFP), peptidase-mitochondrial processing data unit (PMPCB), and upper zone of growth plate and cartilage matrix associated (UCMA), which might be used to monitor the health status of neonatal donkeys. In functional analysis, some differentially expressed proteins were identified related to immune system pathways, which might provide more insight in the immature immunity of neonatal donkeys. To the best of our knowledge, this is the first time to report donkey urinary proteome and our results might provide reference for urinary biomarker discovery used to monitor and evaluate health status of neonatal donkeys.