Mascot was set up to search against the human UniprotKB database (20,319 entries; version 2011_08) assuming trypsin as the digestion enzyme with a maximum of 1 missed cleavage allowed. expression in glycoprotein synthesis. Cellular detoxification machinery may be transformed as well, thereby accommodating increased metabolic activities in late lactation. The multiple developing functions of HM proteins and the corresponding mammary adaption become more apparent from this study. Keywords: human milk, milk proteins, immunoglobulins, match system, cellular processes 1. Introduction The majority of proteins found in human milk are secreted from mammary epithelial cells, though in addition, proteins may cross the mammary epithelial barrier into milk from either maternal blood or the extracellullar matrix (ECM) underlying the mammary epithelium (Physique 1a) [1]. Improved understanding of the lactating mammary gland may provide novel insights into the changes in milk composition and function during feeding. Considerable progress has been made in understanding the lactating mammary gland of ruminants and milk secretion in the past several decades [2]. Functional mammary genomic and transcriptome studies have been carried out for several farm animal species, including the goat mammary transcriptome [3], bovine mammary transcriptome [4,5] and cattle genome [6]. Comparable studies in humans are scarce, which can be ascribed to the absence of non-invasive approaches for examining the human mammary gland during breastfeeding and the lack of cultured cell lines able to secrete milk for studies [7]. Open in a separate window Physique 1 Quantitative analysis of human milk proteome during the first 12 months of lactation. (a) Milk proteome comprises proteins synthesized within the mammary gland (blue circles), as well as proteins either passing through blood circulation (reddish circles and reddish diamonds) or released from mammary extracellular matrix (green triangles); (b) Experimental design: milk whey proteins at six different lactation stages were extracted, digested, labeled, fractionated and analyzed with mass spectrometry as explained in the Experimental Section. Proteins and other nutrient species in breast milk promote the health, growth and development of infants in many ways in addition to simple nutrition [8,9,10,11,12,13,14,15]. Despite the increased understanding of milk functionality, there is little information available regarding the development of milk function over the course of lactation where milk may contribute differently during the development from a newborn to a more mature infant. In an effort to further explore the benefits that human milk can provide, numerous studies have been carried out to investigate the composition and functionality of milk proteins [16,17,18,19,20,21,22,23,24,25]. Bretylium tosylate Our initial comparison of human milk proteins between two lactational stages (1 week and 3 months postpartum) expanded the number of proteins identified and exhibited quantitative differences between the two stages [26]. However, the two lactational time points can provide only a rudimentary depiction that is inadequate to fully understand the developing milk biology and function. For example, Goldman for 60 min) so that samples experienced a pellet of casein micelles on the bottom, a fat layer on the top, and delipidated whey supernatant in the middle. To obtain protein samples, the whey layer was filtered using a 10 kDa molecular-weight cut-off device (Millipore, Billerica, MA, USA) and subjected to buffer exchange with water. 2.3. In-Solution Tryptic Digestion, Isobaric TMT Labeling and Rabbit polyclonal to AFF3 ERLIC Fractionation Protein concentration for filtered whey samples was decided with Dumas combustion methodology using an FP-2000 analyzer (LECO, St. Joseph, MI, USA). 100 g of protein was removed from each sample for reduction and alkylation, followed by tryptic digestion, isobaric tagging, quenching of unreacted TMT reagents and peptide pooling according to the TMT protocol (TMT 6-plex Bretylium tosylate Isobaric Label Reagent Set, Thermo-Fisher Scientific, San Jose, CA, USA) with the following modification in protein precipitation: after protein alkylation, buffer exchange with 100 mM TEAB was Bretylium tosylate used as a replacement step for immediately cold-acetone precipitation. For each of the 10 donors, milk samples collected at 0, 1, 3, 6,.