Analysis Of N-and O-glycomics In Different Developmental Stages Of Drosophila Melanogaster By LC-ESI-MS/MS

Drosophila melanogaster is an important model organism with the advantages of short development cycle,strong fertility,easy feeding,sensitive response and clearly genetic background,which has an irreplaceable place in various fields of biology.Studies have shown that the glycans of Drosophila has similar structures and functions to that of vertebrates in development and disease mechanisms.Therefore,the analysis of Drosophila protein glycosylation has important value for the development of vertebrates,especially for human development and related disease mechanisms.However,reports have been limited to the investigation of certain functional protein glycosylation in Drosophila,none research have been reported on glycomics at different developmental stages of Drosophila.The relationship between the characteristics of glycomics and the development stage in Drosophila is not yet clear,therefore,further studies are anxiously expected to provide a theoretical basis for studying the relationship between human development and protein glycosylation using Drosophila as a model.In this study,N-glycans were released by PNGase F and O-glycans were produced by the use of "one-pot method" with NaOH-PMP system.Electrospray ionization mass spectrometry(ESI-MS)and on-line mass spectrometry(HPLC-MS)analysis methods were deployed to qualitatively and quantitatively analyze N-and O-glycans harvested from three stages as larvae,pupa and adult stages of Drosophila melanogaster.Moreover,N-and O-glycans released from total protein in embryos stage of Drosophila were specific qualitatively and quantitatively analysed by the techniques.These results revealved the characteristics and differences of glycoproteomics at different developmental stages of Drosophila.1.Qualitative and quantitative analysis of N-and O-glycans at Drosophila different developmental stages.The results showed that in the process of post embryonic development,the high mannose-type N-glycans in Drosophila melanogaster decreased significantly,while the expression level of core fucosylated N-glycans increased dramastically.At the same time,the expression level of Tn antigen and Gal1?3GalNAc in core type 1 decreased,while the expression level of Gal1?6GalNAc in O-glycans increase.in the expression level of Gal1?GalNAc in core O-glycans indicated that the core fucosylated N-glycans and core O-glycans Gal1GalNAc may be Play a certain significant role in the development process.It was shown that the complex N-glycans,especially the core fucosylated N-glycans and the core 1-type O-glycans Gal1GalNAc,may have a vital role in embryonic development.2.Qualitative and quantitative analysis of N-and O-glycomics of Drosophila embryo.The results indicate that there are 19 N-glycans in Drosophila embryos,most of which were complex N-glycans.There are many kinds of complex N-glycans,but the percentage of which is low.Three kinds of glycans of single fucosylated and 2 di-fucosylated N-glycans were detected respectively,and there are isomers in four N-glycans.Furthermore,8 species of Mucin O-glycocans in Drosophila embryos are detected,including Tn antigen,corel O-glycan Gal1GalNAc1,and single fucosylated O-glycan.The results show that complex N-glycans and core1 O-glycan Gal1GalNAc1 tightly relate to development and differentiation.3,Analysis of N-and O-glycans in Drosophila embryos by SDS-PAGE.8 protein bands are separated by SDS-PAGE from the total protein of Drosophila embryos,the MW of which are 300 kDa,70 kDa,67 kDa,60 kDa,55 kDa,47 kDa,35 kD,and 24 kDa respectively.N-glycans and O-glycans released from recovered bands are analyzed.The results show that five protein bands contain an high mannose-type N-glycan Man2GlcNAc2,five protein bands contain three N-glycans as Man3GlcNAc2Fuc,Man3GlcNAc5Fuc and Man5GlcNAc2.All eight protein bands contain the Tn antigen GalNAc,7 of which contain core type 1 O-glycans Gal1?6GalNAc,two protein bands contain core type 1 O-glycans Gal1 3GalNAc,and one band contains Fucosylated O-glycans FucHex3HexNAc2,and one band contains the O-glycan of GlcNAcGalNAc,two bands contains the O-glycan of HexHexNAc4.In summary,it is speculated that the Tn antigen GlcNAc and the core type 1 O-glycan Gal1?6GlcNAc play an important role in embryonic development.In addition,high-level expression of GlcNAc1 and Gal1?6GlcNAc implies that band 1(about 300KDa)has certain vital roles of embryonic development.