尿液蛋白富集方法的比较

Comparison of methods for enriching urine proteins

摘要:

人尿液中蛋白含量低，在进行质谱分析时易被高丰度蛋白掩盖。因此，发展高效和高选择性的富集方法，是实现尿蛋白标记物深度覆盖的必要前提。探究不同实验方法对尿液蛋白富集和尿蛋白质组的影响尤为重要。本研究采用超滤法、硝酸纤维素膜富集法和饱和硫酸铵沉淀法，等体积各处理5例健康志愿者和膀胱癌患者10 mL尿液样本，富集尿液蛋白，SDS-PAGE分离尿蛋白，比较不同方法纯化的效率；通过质谱分析，比较不同纯化方法的肽段鉴定效果，确定针对尿液蛋白质组蛋白的最佳富集方法。相对于超滤和硝酸纤维素膜富集法，饱和硫酸铵沉淀法成功地应用于健康人尿蛋白的富集和质谱检测，在保证回收蛋白质量的前提下，可减少高丰度白蛋白的干扰，富集更多低丰度蛋白，提高了质谱鉴定的灵敏度。综上所述，饱和硫酸铵提取尿蛋白的效果较好，该方法具有大规模处理尿液、提高蛋白质组学筛选临床诊断标记物研究的应用潜力。

Abstract:

The abundance of proteins in human urine is low and easily to be masked by high-abundance proteins during mass spectrometry analysis. Development of efficient and highly selective enrichment methods is therefore a prerequisite for achieving deep coverage of urine protein markers. Notably, different experimental methods would affect the urine protein enrichment efficacy and the coverage of urine proteome. In this study, ultrafiltration, nitrocellulose membrane enrichment and saturated ammonium sulfate precipitation were used to process 10 mL urine samples from five healthy volunteers and five bladder cancer patients. The urine proteins were enriched and separate by SDS-PAGE to compare the purification efficiency of different methods. Moreover, the peptide identification effects of different purification methods were analyzed by mass spectrometry to determine the best method for enriching urine protein histones. Saturated ammonium sulfate precipitation method outperformed the ultrafiltration and the nitrocellulose membrane enrichment methods in terms of the protein enrichment efficacy and quality. The interference of highly abundant albumin was reduced, whereas the amount of low-abundance protein was increased, and the sensitivity of mass spectrometry identification was increased. The saturated ammonium sulfate precipitation method may be applied for large-scale urine processing for screening clinical diagnostic markers through proteomics.