人尿液N-糖蛋白/N-糖肽规模化富集鉴定

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

蛋白质的N-糖基化是真核细胞中一种重要的翻译后修饰,N-糖基化修饰在调控细胞黏附、迁移、信号转导及细胞凋亡等方面扮演着关键角色。蛋白质糖基化修饰的异常变化与多种重要疾病的发生相关。尿液具有蛋白质组复杂程度低和非入侵性等特点,适合大量及连续多时间点采样研究。但由于个体差异和生理条件的影响,尿蛋白丰度的生理波动较大。目前缺乏对健康人群尿液N-糖蛋白的个体差异和生理波动的专门性研究,以及生理丰度范围的构建,难以将个体差异、正常生理波动和疾病导致的变化进行有效区分,对疾病标志物研究提出很大挑战。本研究以亲水相互作用色谱法(HILIC)为基础,对该富集方法中活化、清洗与洗脱过程进行优化,其中主要对HILIC填料粒径和富集缓冲体系进行优化,并考察了不同实验条件下N-糖肽富集的鉴定数量、选择性与稳定性,发现当HILIC填料粒径为5μm,在三氟乙酸富集体系下有更高的N-糖蛋白/N-糖肽鉴定水平。在此基础上,对20例健康男性志愿者和20例健康女性志愿者的尿液N-糖蛋白/N-糖肽进行了富集和定性、定量及功能分析。从40例尿液样本中共鉴定到1016个N-糖蛋白、2192条N-糖肽。采用非标定量策略对尿液N-糖肽的生理丰度波动范围进行了考察,尿液N-糖肽的丰度跨度约5个数量级。在此之后探索了健康人群尿蛋白N-糖基化水平的性别差异,筛选出性别相关的差异N-糖蛋白后进行了功能分析。统计学分析显示在尿液样本中性别可能是产生个体差异的重要因素。该工作为基于尿液糖蛋白质组学的功能与机制研究和临床生物标志物筛选提供了有力支撑。

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.