Glycoproteome Study Of Human Plasma And Exosomes

Protein glycosylation is one of the most important post-translational modification.Glycosylation participates in nearly all the biological process,including immune recognition,signal transduction and cell adhesion.There are four types of glycosylation: N-glycosylation,O-glycosylation,C-glycosylation and GPI-anchored glycosylation.Nowadays,most researches focused on N-glycosylation and O-glycosylation.Glycosylation in different biological samples is related with proteins and the physiological environment.Aberrant abundance and structure of glycosylation are closely related with some severe diseases such as cancers.In this issue,we dedicated to developing glycosylation analysis methods for various biological samples.In the view of sialylation and endogenous intact glycopeptides in serum,the correlation of exosome proteins from urine and plasma,and the quantification analysis of urine exosome glycoproteins,the work was carried out in different aspects,such as extraction,enrichment and identification.The results obtained were the first large-scale identifications on endogenous intact glycopeptides.To solve the problems of low abundant and low enrichment specificity of sialylated glycopeptides from serum,we synthesized microspheres modified with multi-histidine,MHM,which could enrich sialylated glycopeptides specially.We obtained a high specificity enrichment of sialylated glycopeptides by making the best of electrostatic interaction,hydrophilic interaction and a synergistic effect between multiple histidines.We enriched 510 intact glycopeptides from initial 4 ?L human serum with over 94.7% specificity of sialylated glycopeptides.It achieved the specific enrichment and analysis of sialylated glycopeptides from complex biological sample.To solve the problem of low abundant and difficulties in data post-processing of endogenous intact glycopeptides from human serum,we developed an integrated platform to work out.We combined heating in acid condition and ultrafiltration to extract endogenous peptides from human serum.Then we utilized hydrophilic interaction chromatography to achieve an efficient enrichment of glycopeptides.The strategies of “twin spectra scheme” and deglycosylation in silico were respectively used to process the data of 223 endogenous intact N-glycopeptides and 51 endogenous intact O-glycopeptides.This is the first analysis of endogenous intact N-glycopeptides and endogenous intact O-glycopeptides from human serum.Through quantitative proteomic technology,we established a correlation of exosome proteins between human urine and plasma.Combined the clinical samples with HCC(Hepatocellular Carcinoma),we obtained proteins with significant difference in patient urine exosomes and plasma exosomes.By comparing the differences between samples from different biological sources,we found 67 proteins existed in urine and plasma exosomes with significant difference in the meanwhile.These proteins had the same or opposite changes in abundance,including kinase LYN,which could regulated oncogene c-MYC expression in HCC cells.It fully demonstrated that there is a correlation of exosome proteins between urine and plasma,which provided a new view for the detection and diagnosis of HCC and even other diseases.At the same time,we analyzed further the patient exosome glycoproteins using quantitative glycosylation proteomics technology.From the perspective of intact glycopeptides with difference,we obtained 81 up-regulated and 103 down-regulated intact glycopeptides from urine exosomes,corresponding 90 glycoproteins with difference.Moreover,39 of these glycoproteins did have no difference in total protein quantitative analysis.Therefore,we conducted quantitative glycoproteomic analysis,which was complementary to the quantitative proteomic analysis,and provided more theoretical support for the clinical diagnosis and mechanism exploration of HCC.