Quantitative Analysis of the Intra- and Inter-Individual Variability of the Normal Urinary Proteome

Urine is a readily and noninvasively obtainable body fluid. Mass spectrometry (MS)-based proteomics has shown that urine contains thousands of proteins. Urine is a potential source of biomarkers for diseases of proximal and distal tissues but it is thought to be more variable than the more commonly used plasma. By LC−MS/MS analysis on an LTQ-Orbitrap without prefractionation we characterized the urinary proteome of seven normal human donors over three consecutive days. Label-free quantification of triplicate single runs covered the urinary proteome to a depth of more than 600 proteins. The median coefficient of variation (cv) of technical replicates was 0.18. Interday variability was markedly higher with a cv of 0.48 and the overall variation of the urinary proteome between individuals was 0.66. Thus technical variability in our data was 7.5%, whereas intrapersonal variability contributed 45.5% and interpersonal variability contributed 47.1% to total variability. Determination of the normal fluctuation of individual urinary proteins should be useful in establishing significance thresholds in biomarker studies. Our data also allowed definition of a common and abundant set of 500 proteins that were readily detectable in all studied individuals. This core urinary proteome has a high proportion of secreted, membrane, and relatively high-molecular weight proteins.

尿液是一种易于获取且无创的体液。基于质谱（Mass Spectrometry, MS）的蛋白质组学研究已证实，尿液中含有数千种蛋白质。尿液可作为近端和远端组织疾病生物标志物的潜在来源，但其变异性被认为高于临床更常用的血浆样本。本研究采用未进行预分级分离的LTQ-Orbitrap平台液相色谱-串联质谱（Liquid Chromatography-Tandem Mass Spectrometry, LC-MS/MS）分析，对7名健康志愿者连续3天的尿液蛋白质组进行了表征。通过对三次独立技术重复上机检测进行无标记定量，本研究实现了对超过600种尿液蛋白质的深度覆盖。技术重复的中位数变异系数（Coefficient of Variation, CV）为0.18。日间变异显著更高，其CV值达0.48；而个体间尿液蛋白质组的总变异系数为0.66。因此，本研究数据中的技术变异占比为7.5%，个体内变异贡献了总变异的45.5%，个体间变异则贡献了47.1%。确定个体尿液蛋白质的正常波动范围，有助于在生物标志物研究中设定显著性阈值。本研究数据还明确了一组在所有受试个体中均可稳定检测到的高丰度共通蛋白质组，共计500种。该核心尿液蛋白质组富含分泌蛋白、膜蛋白以及相对高分子量的蛋白质。