铜螯合纳米磁珠结合MALDI-TOF／TOF MS分析人尿液中多肽组分的方法初探

目的：利用铜螯合纳米磁珠（MB-IMAC-Cu^2＋）系统联合基质辅助激光解析电离-飞行时间质谱（MALDI-TOF／TOFMS）建立一套稳定、可行的技术路线，对尿液中相对分子质量（Mr）〈10000的多肽进行系统研究，包括从样品的采集、存放、提取、检测在内的各个步骤。方法：分析了8名健康男性和8名健康女性的尿液标本。针对样本采集、存放、不同提取方法以及实验整个系统稳定性的问题设计不同组，分别检测各组尿液多肽谱并对结果进行评价。结果：在本系统中，观察到健康人尿液Mr〈10000范围内平均有85个信号峰，不同性别组间尿液多肽谱未见明显差异。以MALDI-TOF／TOF检测得到的出峰情况来评价各种可能因素对实验体系的影响，铜螯合纳米磁珠（MB-IMAC-Cu^2＋）提取多肽效果比较满意；整个实验体系稳定、可靠，重复性好，选择其中所观察的10个峰，变异系数均在10％以内，具有良好的可操作性；室温或者4℃保存最好不超过12h；5次以内有限冻融对出峰没有影响。结论：本实验所探索、建立的方法为下一步研究尿液多肽组学提供了参考。

A method profiling of human urine peptides by MB-IMAC-Cu2+ with MALDI-TOF/TOF MS

Objective：To explore a standardized profiling of urine peptides in sample collection, preparation and detection by magnetic bead separation （ MB-IMAC-Cu^2＋ ） and matrix-assisted laser desorption/ionization time-of-flight （ MALDI- TOF/TOF） mass spectrometry （MS）. Methods：Urine samples from 8 male and 8 female healthy volunteers were investigated. The methods of peptides extraction, sample collection and conservation were directly analyzed by evaluating the results of mass spectrometry. Results：Through MALDI-TOF/TOF MS detection, 85 signal peaks of proteins under the relative molecular weights 10 000 were detected in healthy volunteers urine. There was no significant difference between different sex groups. MB-IMAC-Cu^2＋ could extract multiple peptides. The whole system was very stable with CV less than 10%. Urine should be kept at room temperature or 4℃ less than 12 hours. Limited freezing and thawing had no influence on urine. Conclusions：This profiling method may provide reference for urine peptidome study.