基于肼化学富集法分析人血浆外泌体中的N-糖基化蛋白质

Hydrazide chemistry based enrichment method for N-glycoprotein analysis from human plasma exosomes

目的对人血浆外泌体中N-糖基化蛋白质的组成和功能进行分析。方法通过超速离心法分离获得健康人血浆外泌体,超声处理获得外泌体蛋白,胰蛋白酶将蛋白酶解为肽段。肼化学法分离富集血浆外泌体中的N-糖基化肽段,液相色谱及高分辨质谱对其进行鉴定,基因本体法对鉴定的N-糖基化蛋白质的生物学功能进行分析。结果获得的外泌体具有外泌体典型的茶托样双层膜结构,形态完整,大小均匀,直径分布在100 nm左右。在1 mL健康人血浆外泌体中共鉴定到252个N-糖基化修饰位点,归属于131个N-糖基化蛋白质。可能在丝氨酸型内肽酶活性、丝氨酸型内肽酶活性抑制及钙离子结合等生命过程中发挥重要作用。其中,67个N-糖基化蛋白质与疾病的发生发展密切相关。结论本研究在人血浆外泌体中共鉴定到131个N-糖基化蛋白质。它们可能参与关键的生命活动及疾病的发生发展。

OBJECTIVE To analyze the composition and function of N-glycoproteins in human plasma exosomes. METHODS Exosomes from human plasma were extracted by ultracentrifugation . The proteins from plasma exosomes were released by ultrasound and the obtained proteins were enzymatically degraded by trypsin to peptides. The N-glycopeptides in the mixture of the digested peptides was enriched by the hydrazide resin. The enriched N-glycoproteins were identified by mass spectrometry and subjected to gene ontology (GO) annotation analysis. RESULTS The plasma exosomes isolated by ultracentrifugation had a typical cup-shaped structure and the particle sizes were approximately 100 nm. Totally, 252 N-glycosylation sites corresponding to 131 N-glycoproteins were enriched and identified from exosomes of 1 mL human plasma digestion, which participated in many important biological processes, such as serine-type endopeptidase activity, serine-type endopeptidedase inhibitor acitivity and calcium ion binding. Sixty-seven of the N-glycoproteins had close relations with the occurrence of diseases. CONCLUSION Totally, 131 N-glycoproteins were identified in the research from the plasma exosome protein. These N-glycoproteins precipitate in many important biological processes and have close correlations with the occurrence and progression of various diseases.