| Human milk is the optimal choice for feeding newborns,but exclusive breastfeeding may not always be feasible,appropriate,or sufficient,necessitating the development of formula products that can meet the nutritional needs of newborns.Donkey milk has remarkable similarities to human milk in terms of organoleptic characteristics and chemical composition,as well as being highly digestible and clinically well tolerated,making it considered a suitable potential alternative to breast milk.During lactation,the synthesis and secretion of milk proteins in the mammary epithelium are accompanied by chemical modifications,of which N-glycosylation is the most prevalent and complex heterogeneous post-translational modification of whey and milk fat globule membrane(MFGM)proteins,with important implications for the physiological structure and biological activity of the protein substrates.However,a systematic study of the site-specific N-glycosylation patterns of human and donkey milk whey and MFGM proteins during lactation has not been investigated.In this study,we systematically mapped the sitespecific N-glycosylation landscape of human and donkey milk whey and MFGM proteins during lactation based on site-specific glycoproteomic strategies,which elucidated the differences in the molecular composition of human and donkey milk whey and MFGM Nglycoproteins and the dynamic changes of their site-specific N-glycosylation modifications during lactation.This study provided a molecular basis for an in-depth investigation of the biological functions of human and donkey milk whey and MFGM N-glycoprotein,as well as a theoretical basis for developing strategies to improve the healthfulness of breast milk and the development of donkey milk-based infant formula products.The main results are as follows:1.Using label-free site-specific glycoproteomics,a total of 1,153 site-specific N-glycans on213 N-glycosites from 138 glycoproteins were identified in human milk whey proteins,with the identified N-glycans being primarily sialylated and fucosylated.Among these,100 sitespecific N-glycans on 22 N-glycosites from 14 glycoproteins were significantly changed in human colostrum(HC)and human mature milk(HM)whey proteins(P-value < 0.05).Over20% of the whey glycoproteins in human milk had more than one N-glycosite,and over 20%of the N-glycosites carried more than one N-glycan.Gene ontology(GO)annotation analysis showed that HC and HM whey N-glycoproteins were mainly enriched in complement activation,classical pathway,extracellular exosome part,and immunoglobulin receptor binding.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis revealed that HC and HM whey N-glycoproteins were mainly involved in the PI3K-Akt signaling pathway.2.Using label-free site-specific glycoproteomics,a total of 3,342 site-specific N-glycans on549 N-glycosites from 173 glycoproteins were identified in human MFGM proteins,with the identified N-glycans being primarily sialylated and fucosylated.Among these,100 site-specific N-glycans on 41 N-glycosites from 27 glycoproteins were significantly changed in HC and HM MFGM proteins(P-value < 0.05).Over 40% of the MFGM glycoproteins in human milk had more than one N-glycosite,and over 80% of the N-glycosites carried more than one N-glycan.GO annotation analysis showed that HC MFGM N-glycoproteins were mainly enriched in cell adhesion,extracellular exosome part,and MHC class II protein complex binding,while HM MFGM N-glycoproteins were mainly enriched in antimicrobial humoral response,extracellular exosome part,and heparin binding.KEGG pathway analysis revealed that HC and HM MFGM N-glycoproteins were mainly involved in the phagosome.3.Using label-free site-specific glycoproteomics,a total of 946 site-specific N-glycans from233 N-glycosites on 164 glycoproteins were identified in donkey milk whey proteins,with the identified N-glycans being primarily fucosylated.Among these,42 site-specific N-glycans from16 N-glycosites on 14 glycoproteins were significantly changed in donkey colostrum(DC)and donkey mature milk(DM)whey proteins(P-value < 0.05).Approximately 25% of the whey glycoproteins in donkey milk had more than one N-glycosite,and over 45% of the N-glycosites carried more than one N-glycan.GO annotation analysis showed that DC whey N-glycoproteins were mainly enriched in positive regulation of B cell activation,extracellular space part,and endopeptidase inhibitor activity,while DM whey N-glycoproteins were mainly enriched in phagocytosis,recognition,extracellular space part,and endopeptidase inhibitor activity.4.Using label-free site-specific glycoproteomics,a total of 1,787 site-specific N-glycans from 509 N-glycosites on 380 glycoproteins were identified in donkey MFGM proteins,with the DC and DM MFGM glycoproteins mainly carrying fucosylated and sialylated N-glycans,respectively.Among these,52 site-specific N-glycans from 30 N-glycosites on 23 glycoproteins were significantly changed in DC and DM MFGM proteins(P-value < 0.05).Over 20% of the MFGM glycoproteins in donkey milk had more than one N-glycosite,and over 45% of the Nglycosites carried more than one N-glycan.GO annotation analysis showed that DC MFGM Nglycoproteins were mainly enriched in cell adhesion,cell surface part,and heparin binding,while DM MFGM N-glycoproteins were mainly enriched in membrane protein ectodomain proteolysis,extracellular space part,and heparin binding.5.An intact glycopeptide-centric glycoproteome work pipeline was used to explore differences in site-specific N-glycosylation between human and donkey milk whey proteins during lactation.The 868,425,628,and 347 site-specific N-glycans from 110,57,117,and 59 glycoproteins were identified in HC,HM,DC,and DM,respectively.The N-glycosites of both human and donkey milk whey N-glycoproteins were located at the N-X-S/T consensus sequence motif,and the N-glycans mainly had pentasaccharide core structures,fucosylated pentasaccharide core structures,as well as high mannose,Glc NAc,and Lac NAc(sialylated)branch structures.GO annotation analysis showed that the four whey N-glycoproteins were mainly enriched in innate immune response,extracellular space part,and antigen binding.KEGG pathway analysis revealed that the four whey N-glycoproteins were mainly involved in immune-related pathways such as complement and coagulation cascades.6.An intact glycopeptide-centric glycoproteome work pipeline was used to explore sitespecific N-glycosylation differences between human and donkey MFGM proteins during lactation.The 2,617,986,1,360,and 457 site-specific N-glycans from 214,196,296,and 77 glycoproteins were identified in HC,HM,DC,and DM MFGM proteins,respectively.The Nglycosites of both human and donkey MFGM N-glycoproteins were located at the N-X-S/T consensus sequence motif,and the N-glycans mainly had pentasaccharide core structures,fucosylated pentasaccharide core structures,as well as high mannose,Glc NAc,and Lac NAc(sialylated)branch structures.GO functional annotation analysis showed that the four MFGM N-glycoproteins were mainly enriched in cell adhesion and plasma membrane part.The primary molecular function of human MFGM N-glycoprotein was protein binding,while that of donkey MFGM N-glycoprotein was calcium ion binding.KEGG pathway analysis revealed that the four MFGM N-glycoproteins were mainly involved in immune-related pathways such as phagosome. |
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