In-depth proteome analysis of more than 12,500 proteins in buffalo mammary epithelial cell line identifies protein signatures for active proliferation and lactation

The mature mammary gland is made up of a network of ducts that terminates in alveoli. The innermost layer of alveoli is surrounded by the differentiated mammary epithelial cells (MECs), which are responsible for milk synthesis and secretion during lactation. However, the MECs are in a state of active proliferation during pregnancy, when they give rise to network like structures in the mammary gland. Buffalo ( Bubalus bubalis ) constitute a major source of milk for human consumption, and the MECs are the major precursor cells which are mainly responsible for their lactation potential. The proteome of MECs defines their functional state and suggests their role in various cellular activities such as proliferation and lactation. To date, the proteome profile of MECs from buffalo origin is not available. In the present study, we have profiled in-depth proteome of in vitro cultured buffalo MECs (BuMECs) during active proliferation using high throughput tandem mass spectrometry (MS). MS analysis identified a total of 8330, 5970, 5289, 4818 proteins in four sub-cellular fractions (SCFs) that included cytosolic (SCF-I), membranous and membranous organelle’s (SCF-II), nuclear (SCF-III), and cytoskeletal (SCF-IV). However, 792 proteins were identified in the conditioned media, which represented the secretome. Altogether, combined analysis of all the five fractions (SCFs- I to IV, and secretome) revealed a total of 12,609 non-redundant proteins. The KEGG analysis suggested that these proteins were associated with 325 molecular pathways. Some of the highly enriched molecular pathways observed were metabolic, MAPK, PI3-AKT, insulin, estrogen, and cGMP-PKG signalling pathway. The newly identified proteins in this study are reported to be involved in NOTCH signalling, transport and secretion processes.