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

Abstract

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.The mammary gland, a unique organ of mammals, is a derivative of ventral skin 1 and has evolved to provide nutrition and immune protection to the offspring. Though the anatomical and physiological features of a mammary gland are diverse, the basic structure is similar in all species. The mature mammary gland is constituted by a network of tubular-alveolar structures, with their lumen lined by a layer of polarized secretory mammary epithelial cells (MECs). The MECs are dynamic components of the mammary gland, which undergo repeated rounds of proliferation and differentiation during different developmental stages such as pregnancy, and lactation 2 . The quantity and functional differentiation of MECs in the udder determine the milk-producing ability in livestock. Bovine (ruminants) are physiologically distinct from other mammals such as humans, mice and rats. The present study on in-depth proteome analysis of buffalo (Bubalus bubalis) MECs (BuMECs) advances our knowledge on the protein machinery operating in mammary gland development and lactogenesis in buffalo, which can be extrapolated in other mammalian species as well. Among the domestic livestock species, cattle (Bos taurus) and

TMT based deep proteome analysis of buffalo mammary epithelial cells and identification of novel protein signatures during lactogenic differentiation

“…These DEPs were suggested to be related to metabolic, binding, and catalytic activities. Proteomic studies of buffalo mammary glands are relatively scarce; however, our group’s recent study identified 12,609 proteins in the in vitro grown proliferating BuMECs, using subcellular fractionation complexed with shotgun proteomics approach 30 …”

“…The pooled labeled peptides were fractionated using the Quaternary UHPLC system (Ultimate 3000, Thermo Scientific, USA) into 24 individual fractions as explained previously by Jaswal et al 30 Briefly, the pooled peptide sample was loaded onto the C18 column (4.6 × 250 mm, 5 µm, Grace, USA) on a Dionex, UHPLC system. A total of 96 time‐based fractions were collected and further pooled into 24 individual fractions by mixing most hydrophobic with most hydrophilic based on concatenation approach (Figure 1B).…”

“…Peak lists were generated by qTOF control (version 24.8) using the Hystar post‐processing program to automatically subtract baseline, smoothen peaks and generate centroid data. The data so generated was searched against the Bos taurus database for identification and quantification of proteins using Mascot (2.4.1 Matrix Science, UK) search engine on Protein Scape software 3.2 (Buker Daltonics, Germany) 30 . Search criteria used were as follows: taxonomy‐ other Mammalia, trypsin digestion, allowing up to one missed cleavage, fixed modification‐ carbamidomethylation at cysteine residue, and TMT labeling at peptide N‐terminus and lysine residue, whereas oxidation at methionine was considered as variable, peptide tolerance of 50 ppm, and MS/MS tolerance of 0.05 Da.…”

Domestic animal proteomics in the 21st century: A global retrospective and viewpoint analysis

“…Domestic animal proteomics has also been applied to animal production systems related to different aspects of lactation. These included the study of lactating mammary epithelial cells in cattle and water buffalo [57,58], bovine milk proteome characterization and differentiation between breeds [59,60] or the establishment of protein biomarkers for early pregnancy diagnosis [61] estrus detection [62], male fertility [63,64].…”

A positive pressure workstation for semi‐automated peptide purification of complex proteomic samples

“…In addition, workstations including solvent dispensing and computer‐controlled pressure gradients are highly valuable in saving time, consumables and costs while maintaining reproducible parallel preparation of up to 96 samples. As several deep proteome investigations have been published recently, 8,9 it is evident that the general need for high‐throughput methods has greatly increased also for proteomic investigations over recent years.…”