Comprehensive Analysis Of The Glycome And Glycoproteome Of Bovine Milk-derived Exosomes

Exosomes are naturally occurring nanosized(30-150 nm)extracellular vesicles that can be released from many types of cells,tissues and biological fluids.Exosomes play a vital role in cell-cell communication by the transfer of various miRNA,mRNA,DNA and proteins.Bovine milk-derived exosomes(BMDEs)are broadly presented in dairy products and easily available.With the high biocompatibility,long circulatory half-life and low immunogenicity,the BMDEs were broadly applied in the targeted drug delivery system.To understand the function of proteins and their post translational modification(PTM)of BMDEs in target delivery,the protein glycosylation analysis of BMDEs is essential.In this study,we developed a set of methods for identification glycome and glycoproteome of BMDEs and bovine milk whey.First,the ultracentrifugation and isoelectric precipitation were used to remove the high abundance protein in the whey and obtain BMDEs from the bovine milk.Subsequently,the N-/O-glycan from exosomes and whey were released by enzymatic and chemical methods,respectively,and modified by different derivatization methods to improve their ionization efficiency and coverage in mass spectrometry.In total of13 free oligosaccharides(FOSs),44 O-glycans and 94 N-glycans were identified in bovine milk.Moreover,two sialylated(F0H5N3S0G1 and F0H4N4S0G1)and Lacdi NAc(F0H5N8S0G0)N-glycans were found to be potential biomarkers for BMDEs,which were only identified in the BMDEs.It showed that the N-glycans in milk are mainly fucosylated and sialylated,which can not only serve as the recognition site of exosomes,promoted the rapidly transfer of drug-coated exosomes,but also resist pathogens in the process of transmission.In addition,we used a one-step method for the glycopeptide separation from the glyco-depleted global peptides,and then all the glycopeptides and peptides were analyzed by HPLC-ESI-MS/MS.A total of 1223 proteins which contained 86 glycoproteins,representing767 unique intact glycopeptides,113 unique glycosite-containing peptides and 80 glycan structures were identified in BMDEs.Approximately 95 glycopeptides were discovered in BMDEs.Moreover,the glycoproteome strategy was applied to analyze the protein glycosylation of bovine,goat and porcine milk.About 136,82 and 125 glycoproteins,and 258,259 and 247 N-glycans were identified from these milks,respectively.Collectively,the fucosylated and sialylated glycosylated proteins in the exosomes were mainly glycoproteins in term of abundance and diversities.These glycoproteins were involved in the complement coagulation cascade,which play an important role in mediating inflammation,regulating immunity and clearing immune complexes.