纳升液相色谱-高分辨串联质谱研究不同分离方法对唾液外泌体蛋白质组的影响

Comparison of Separation Methods for Isolation in Analysis of Human Saliva-derived Exosomes Proteomics by Nano-liquid Chromatography-Mass Spectrometry

外泌体是一系列胞外囊泡,是生物标志物的来源,但目前尚无灵敏高效的唾液外泌体蛋白质分离方法。本研究采用质谱方法比较唾液外泌体试剂盒(Exo Quick,EQ)方法和超高速离心(Ultracentrifugation,UC)方法分离外泌体的效果以及尿素缓冲液、RIPA裂解液、SDS裂解液提取外泌体蛋白质的效果。Brodford法和BCA定量测定结果表明,EQ方法分离0.5 m L唾液外泌体得到的蛋白质含量高于UC方法分离2 m L唾液所得到的蛋白质。进一步的质谱分析表明,前者鉴定到的蛋白质数目亦多于后者;试剂盒分离唾液外泌体与尿素缓冲液提取外泌体蛋白质的方法联用效果最佳,鉴定到194种蛋白质。本方法重现性好、样品用量少、检测结果稳定,可为在唾液外泌体中筛选疾病的标志物提供方法学参考。

Exosomes are a series of extracellular vesicles,which are the source of biomarkers.However,there are no sensitive and efficient methods for the isolation of saliva exosome proteins.In this work,we explored the effect of isolation of saliva exosome by ExoQuick(EQ)and ultracentrifugation(UC)method,and the effect of UREA buffer,RIPA lysate,and SDS lysate on exosome protein extraction by nano-liquidchromatography-high-resolution tandem mass spectrometry.The quantitative results of Brodford method showed that the protein content of exosomes obtained by EQ method from 0.5 mL of saliva was higher than that by UC method from 2 mL of saliva.Further mass spectrometry analysis showed that the former identified more proteins than the latter.The effect of the combination of using EQ method and UREA buffer to obtain saliva exosomes proteins was the best.With this method,a total of 194 proteins were identified.The method had good reproducibility,low sample requirement,and stable mass spectrometry detection,and could provide methodological support for finding markers of disease in saliva exosomes.