[Large-scale enrichment and identification of human urinary N-glycoproteins/N-glycopeptides]

N-Glycosylation of proteins, an important post-translational modification in eukaryotic cells, plays an essential role in the regulation of cell adhesion, migration, signal transduction, and apoptosis. Abnormal changes in protein glycosylation are closely related to the occurrence of many critical diseases, including diabetes, tumors, and neurological, kidney, and inflammatory diseases. A non-invasive type of liquid biopsy, urine sampling has the advantage of reducing the complexity of proteomic analysis. This facilitates the design of large-scale and continuous or multi-time point sampling strategies. However, the dynamic range of urinary protein abundance is relatively large, owing to individual differences and physiological conditions. Currently, there is a lack of specialized research on individual differences, physiological fluctuations, and physiological abundance ranges of urinary N-glycoproteins in large healthy populations. Therefore, it is difficult to accurately distinguish individual differences and normal physiological fluctuations from changes caused by disease; this poses a great challenge in disease marker research. Liquid chromatography-mass spectrometry (LC-MS) is an analytical technique widely used for the large-scale profiling of proteomes in biological systems, and the enrichment of N-glycopeptides is a prerequisite for their detection by MS.In this study, we established an approach based on hydrophilic interaction chromatography (HILIC) by optimizing the activation, cleaning, and elution processes of the enrichment method, for instance through the optimization of particle size and solvent composition, and investigated the identification number, selectivity, and stability of N-glycoprotein/N-glycopeptide enrichment under different experimental conditions. We found that N-glycoproteins and N-glycopeptides were highly enriched in a trifluoroacetic acid system with 5-μm filling particles in the HILIC column. On this basis, we analyzed the levels of N-glycoproteins/N-glycopeptides in urine samples. The consistency of N-glycoprotein/N-glycopeptide levels in urine samples taken from the same healthy person for five consecutive days was investigated by correlation analysis. This analysis revealed that the urinary N-glycoproteome of the same healthy person was relatively stable over a short period of time. Next, urinary samples from 20 healthy male volunteers and 20 healthy female volunteers were enriched for N-glycoproteins/N-glycopeptides, which were profiled by MS through qualitative and quantitative analyses. Screening and functional analysis of differential proteins were then carried out. A total of 1016 N-glycoproteins and 2192 N-glycopeptides were identified in the mid-morning urine samples of the 40 healthy volunteers. A label-free quantitation strategy was used to investigate the fluctuation range of the physiologically abundant urinary N-glycopeptides. The abundance of urinary N-glycopeptides spanned across approximately five orders of magnitude. Subsequently, gender differences in the N-glycosylation levels of urinary proteins were also explored in healthy people. Functional analysis of the N-glycoproteins that exhibited gender differences in abundance was performed. Based on multivariate statistical analysis, 206 differentially expressed proteins (p<0.05, fold change (FC)> 4) were identified. In females, we found 175 significantly down-regulated N-glycoproteins and 31 significantly up-regulated N-glycoproteins with respect to males. The expression levels of N-glycopeptides between the two groups suggested a clear gender difference. To investigate the biological processes and functions of these proteins, gene ontology (GO) analysis was performed on the N-glycoproteins/N-glycopeptides differentially expressed between males and females. Metabolic pathway analysis was also carried out based on the kyoto encyclopedia of genes and genomes (KEGG). Differentially expressed N-glycoproteins were mostly associated with platelet degranulation, extracellular region, and ossification. The top three relevant pathways were glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, and lipid metabolism. Overall, sex may be an important factor for urinary N-glycoproteome differences among normal individuals and should be considered in clinical applications. This study provides relevant information regarding the function and mechanisms of the urinary glycoproteome and the screening of clinical biomarkers.