Ghrelin is a hormone produced mainly by the cells lining the gastric mucosa. Ghrelin was first extracted from human and rat stomachs, and identified as an endogenous stimulator of growth hormone release. Ghrelin is synthesized and produced in several tissues, but the gastric mucosa remains the major source of circulating ghrelin. Besides growth hormone release, ghrelin stimulates appetite and plays some major roles in different organs. In several studies, ghrelin was described as a hormone with multiple functions and diverse biological actions. Ghrelin exists in two major forms, active ghrelin and inactive ghrelin, and only the active from binds to the receptor. The majority of total circulating ghrelin is inactive ghrelin with no identified function. The aims of the present study were to measure active and total ghrelin in dairy cow’s milk and plasma during early lactation, and to observe changes in the ghrelin concentrations over time. We are interested in this period of time since the milk during early lactation contains a variety of biologically active hormones that are vital for newborn calves. In this study, fifteen Holstein dairy cows were selected randomly from different lactations. Milk and blood samples were taken daily from cows at early lactation for 10 days, and from some cows in mid-lactation. A laboratory test was used to measure active and total ghrelin in milk and plasma samples. Supplementary measurements such as milk fat, lactose, protein, and milk yield were recorded. Active and total milk ghrelin concentrations were found to be significantly higher in the first day of lactation during colostrum production. Interestingly, the percentage of active to total ghrelin in milk and blood was constant in all days tested, suggesting that this constant percentage can be used to estimate active or total ghrelin, if one of them is know, from the same sample. However, no correlation was observed between the percentage of milk ghrelin and plasma ghrelin or with other milk components. In conclusion, the presence of ghrelin in colostrum and milk in measurable amounts of both active and total form suggests that it is a critical compound for the metabolic activity of newborn calves and functions transiently to regulate the activity of some physiological processes until the endocrine system of the new calves starts to function independently.

Sample preparation is the most critical step in proteomics as it directly affects the subset of proteins and peptides that can be reliably identified and quantified. Although a variety of efficient and reproducible sample preparation strategies have been developed, their applicability and efficacy depends much on the biological sample. Here, three approaches were evaluated for the human milk and plasma proteomes. Protein extracts were digested either in an ultrafiltration unit (filter-aided sample preparation, FASP) or in-solution (ISD). ISD samples were desalted by solid-phase extraction prior to nRPC-ESI-MS/MS. Additionally, milk and plasma samples were directly digested by FASP without prior protein precipitation. Each strategy provided inherent advantages and disadvantages for milk and plasma. FASP appeared to be the most time efficient procedure with a low miscleavage rate when used for a biological sample aliquot, but quantitation was less reproducible. A prior protein precipitation step improved the quantitation by FASP due to significantly higher peak areas for plasma and a much better reproducibility for milk. Moreover, the miscleavage rate for milk, the identification rate for plasma, and the carbamidomethylation efficiency were improved. In contrast, ISD of both milk and plasma resulted in higher miscleavage rates and is therefore less suitable for targeted proteomics.