Regulatory role of phosphoproteins in the development of bovine small intestine during early life.

The small intestine is the primary site of nutrient digestion and absorption, which plays a key role in the survival of neonatal calves. A comprehensive assessment of the phosphoproteomic changes in the small intestine of neonatal calves is unavailable; therefore, we used phosphopeptide enrichment coupled with liquid chromatography-tandem mass spectrometry to investigate the changes in the phosphoproteome profile in the bovine small intestine during the first 36 h of life. Twelve neonatal male calves were assigned to one of the following groups: (1) calves not fed colostrum and slaughtered approximately 2 h postpartum (n = 3), (2) calves fed colostrum at 1 to 2 h and slaughtered 8 h postpartum (n = 3), (3) calves fed 2 colostrum meals (at 1-2 and 10-12 h) and slaughtered 24 h postpartum (n = 3), (4) calves fed 3 colostrum meals (at 1-2, 10-12, and 22-24 h) and slaughtered 36 h postpartum (n = 3). Mid-duodenal, jejunal, and ileal samples of the calves were collected after slaughter. We identified 1,678 phosphoproteins with approximately 3,080 phosphosites, which were mainly Ser (89.9%), Thr (9.8%), and Tyr (0.3%) residues; they belonged to the prodirected (52.9%), basic (20.4%), acidic (16.6%), and Tyr-directed (1.7%) motif categories. The regional differentially expressed phosphoproteins included zonula occludens 2, sorting nexin 12, and protein kinase C, which are mainly associated with developmental processes, intracellular transport, vesicle-mediated transport, and immune system process. They are enriched in the endocytosis, tight junction, insulin signaling, and focal adhesion pathways. The temporal differentially expressed phosphoproteins included occludin, epsin 1, and bridging integrator 1, which were mainly associated with macromolecule metabolic process, cell adhesion, and growth. They were enriched in the spliceosomes, adherens junctions, and tight junctions. The observed changes in the phosphoproteins in the tissues of small intestine suggest the protein phosphorylation plays an important role in nutrient transport and immune response of calves during early life, which needs to be confirmed in a larger study.

Quantitative Phosphoproteomics of Milk Fat Globule Membrane in Human Colostrum and Mature Milk: New Insights into Changes in Protein Phosphorylation during Lactation.

Phosphorylation is a widespread posttranslational protein modification and is important in various biological processes. However, milk fat globule membrane (MFGM) phosphoproteins have not been explored systematically in human milk. Here, we used quantitative phosphoproteomics to analyze phosphorylation sites in human MFGM proteins and their differences at different stages of lactation; 305 phosphorylation sites on 170 proteins and 269 phosphorylation sites on 170 proteins were identified in colostrum and mature MFGM, respectively. Among these, 71 phosphorylation sites on 48 proteins were differentially expressed between the different stages of lactation. Osteopontin in human MFGM was the most heavily phosphorylated protein, with a total of 39 identified phosphorylation sites. Our results shed light on phosphorylation sites, composition, and biological functions of MFGM phosphoproteins in human colostrum and mature milk, and provide novel insights into the crucial roles of protein phosphorylation during infant development.