Preparation Of Novel Hydrophilic Enriched Materials And Identification Of Normal Human Urine Glycosylation Protein

Objectives 1 Protein N-glycosylation is involved in many important biological processes and currently is a hotspot in the study of diagnostic markers for many major human diseases.Compared with other liquid biopsy samples,urine is noninvasive,mass-produced and is not regulated by steady-state,therefore,it may reflects the biological and pathological state of the whole body.Large-scale and in-depth identification of human urine protein N-glycosylation is of great significance for biomarker screening.2 Because of the low abundance of N-glycoprotein in human urine and,modification stoichiometry and microheterogeneity of N-glycosylation,it is a challenging task for N-glycopeptide identification by mass spectrometry.Methods 1 The peptides from 30 cases of healthy human urine protein were enriched by hydrophilic material,and the enriched peptide was identified by mass spectrometry.The obtained mass spectrometry data was introduced into the analysis software to obtain the glycopeptide and the glycosylated site information;2 Based on the reaction of "thiol-ene" monomers between [3-(methacrylamido)propyl] dimethyl(3-thiopropyl)ammonium hydroxide(SPP)and mercapto silicon dioxide,a novel zwitterionic stationary phase with high hydrophilicity.Results 1 Male and female were identified 946 and 880 glycoproteins,bioinformatics analysis to obtain healthy men and women of the GO analysis of information is consistent;2 SPP-silica was successfully applied to the HILIC enrichment and mass spectrometry identification of urinary protein N-glycopeptide from healthy adults.The same sample was enriched by three batches of SPP-silica and analyzed by mass spectrometry.959,837 and 877 N-glycosylation sites were identified,respectively,with selectivity of more than 70%.A total of 1065 N-glycosylation sites in 633 urinary glycoproteins was identified,which is 12.2% larger than that reported in the literature.81.2% and 73.9% of the obtained Nglycoproteins and sites were identified in at least two experiments.The above results demonstrate the application potential of this hydrophilic material in urinary glycoproteomics research and biomarker screening.Conclusions 1 A stable and efficient method for large-scale quantitative analysis of urine glycosylated proteins was successfully established,and the establishment of a large-scale healthy human urine protein glycosylation modification data set,in order to achieve the screening of potential biomarkers to lay the foundation;2 Synthesis of new zwitterionic hydrophilic enrichment materials,and used in complex samples,the efficiency of the enrichment of the peptide was significantly improved.