Abstract
BackgroundProtein phosphorylation plays an important role in lactation. Differentially modified phosphorylation sites and phosphorylated proteins between peak lactation (PL, 90 days postpartum) and late lactation (LL, 280 days postpartum) were investigated using an integrated approach, namely, liquid chromatography with tandem mass spectrometry (LC-MS/MS) and tandem mass tag (TMT) labeling, to determine the molecular changes in the mammary tissues during the different stages of goat lactation.ResultsA total of 1,938 (1,111 upregulated, 827 downregulated) differentially modified phosphorylation sites of 1,172 proteins were identified (P values < 0.05 and fold change of phosphorylation ratios > 1.5). Multiple phosphorylation sites of FASN, ACACA, mTOR, PRKAA, IRS1, RPS6KB, EIF4EBP1, JUN, and TSC2 were different in PL compared with LL. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the calcium signaling pathway, oxytocin signaling pathway and MAPK signaling pathway were enriched. The western blot results showed that the phosphorylation levels of ACACA (Ser80), EIF4EBP1 (Thr46) and IRS1 (Ser312) increased and JUN (Ser63) decreased in PL compared with LL. These results were consistent with the phosphoproteome results.ConclusionsIn this study, we identified for the first time the differentially modified phosphorylation sites in goat mammary tissues between PL and LL. These results indicate that the multiple differentially modified phosphorylation sites of FASN, ACACA, mTOR, PRKAA, IRS1, RPS6KB, EIF4EBP1, TSC2, and JUN and proteins involved in the calcium signaling pathway, oxytocin signaling pathway, and MAPK signaling pathway are worthy of further exploration.
Highlights
Protein phosphorylation plays an important role in lactation
Sample repeatability test and mass spectrometry quality control Differentially modified phosphorylation sites and phosphorylated proteins from the PL and LL of goats were analyzed by LC–MS/MS and tandem mass tag (TMT)
We found for the first time that there were multiple differentially modified phosphorylation sites between PL and LL, including Fatty acid synthase (FASN), Acetyl-CoA carboxylase isoform X3 (ACACA) (29, 80, 1251 and 25), Serine/threonine-protein kinase mTOR (mTOR) (1261), 5′-AMPactivated protein kinase catalytic subunit alpha-2 (PRKAA) (377 and 395), Insulin receptor substrate 1 (IRS1) (629, 312, 1138 and 1070), Ribosomal protein S6 kinase beta-1 isoform X1 (RPS6KB) (374 and 394), EIF4EBP1 (65) and TSC2 (1719, 1327 and 918)
Summary
Protein phosphorylation plays an important role in lactation. Phosphorylation is one of the important posttranslational modifications of proteins; it is related to many activities of life, such as signal transduction, gene expression, cell cycle and cell apoptosis [1]. Dynamic changes in protein phosphorylation occur in goat mammary epithelial cells during the PL period and LL period [3, 4]. Previous studies have shown that protein phosphorylation regulates lactation in animals [5, 6]. Phosphoproteome analysis was conducted on the oocytes of goats [7] and the heart and liver of mice [8, 9], phosphorylated proteomic analysis has not been performed in the PL and LL stages of dairy goat mammary tissues. The regulatory mechanism of phosphorylated proteins during lactation is not well known
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