Quantitative Proteome Profiling of Normal Human Circulating Microparticles

Circulating microparticles (MPs) are produced as part of normal physiology. Their numbers, origin, and composition change in pathology. Despite this, the normal MP proteome has not yet been characterized with standardized high-resolution methods. We here quantitatively profile the normal MP proteome using nano-LC–MS/MS on an LTQ-Orbitrap with optimized sample collection, preparation, and analysis of 12 different normal samples. Analytical and procedural variation were estimated in triply processed samples analyzed in triplicate from two different donors. Label-free quantitation was validated by the correlation of cytoskeletal protein intensities with MP numbers obtained by flow cytometry. Finally, the validity of using pooled samples was evaluated using overlap protein identification numbers and multivariate data analysis. Using conservative parameters, 536 different unique proteins were quantitated. Of these, 334 (63%) were present in all samples and represent an MP core proteome. Technical triplicates showed <10% variation in intensity within a dynamic range of almost 5 decades. Differences due to variable MP numbers and losses during preparative steps could be normalized using cytoskeletal MP protein intensities. Our results establish a reproducible LC–MS/MS procedure, provide a simple and robust MP preparation method, and yield a baseline MP proteome for future studies of MPs in health and disease.循环微颗粒（microparticles, MPs）是正常生理过程的天然产物，其数量、来源与组成在病理状态下会发生显著改变。尽管如此，目前尚未有研究采用标准化高分辨方法对正常MP蛋白质组开展系统性表征。本研究依托优化后的样品收集、制备与分析流程，借助搭载LTQ-Orbitrap分析仪的纳米液相色谱-串联质谱（nano-LC–MS/MS），对12份不同正常样本中的MP蛋白质组进行了定量表征。我们通过对两名不同供体的样本进行三次重复处理与三次重复分析，评估了分析方法与实验流程带来的变异；通过流式细胞术获取的MP计数与细胞骨架蛋白强度的相关性，验证了无标记定量方法的有效性；最终通过重叠蛋白鉴定数量与多变量数据分析，评估了使用混合样品的合理性。采用保守参数设置后，本研究共定量到536种不同的独特蛋白质，其中334种（占比63%）在所有样本中均有检出，构成了MP核心蛋白质组。技术重复实验显示，在近5个数量级的动态范围内，蛋白强度的变异程度均低于10%。针对MP数量差异及样品制备步骤中的损失所导致的定量差异，可通过细胞骨架MP蛋白的强度进行标准化校正。本研究建立了一套可重复的LC–MS/MS实验流程，提供了一种简便且稳健的MP制备方法，并获得了可用于后续健康与疾病状态下MP研究的基线MP蛋白质组数据集。