Exploration of the lactation function of protein phosphorylation sites in goat mammary tissues by phosphoproteome...

Protein 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.

A 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.

In 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.