Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark.
J Proteome Res. 2012 Apr 6;11(4):2154-63. doi: 10.1021/pr200901p. Epub 2012 Mar 1.
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 (MPs) 是正常生理过程的一部分产生的。它们的数量、来源和组成在病理学中发生变化。尽管如此，正常 MPs 的蛋白质组尚未通过标准化的高分辨率方法进行描述。我们在这里使用纳升液相色谱-串联质谱 (nano-LC-MS/MS) 在 LTQ-Orbitrap 上对 12 个不同的正常样本进行优化的样本收集、制备和分析，对正常 MPs 的蛋白质组进行了定量分析。对来自两个不同供体的三重处理样本进行三重分析，以评估分析和程序变化。通过与通过流式细胞术获得的 MPs 数量相关的细胞骨架蛋白强度的相关性，验证了无标记定量的有效性。最后，使用重叠蛋白鉴定数量和多变量数据分析来评估使用混合样本的有效性。使用保守的参数，定量了 536 种不同的独特蛋白。其中，334 种（63%）存在于所有样本中，代表 MPs 的核心蛋白质组。在近 5 个数量级的动态范围内，技术重复的强度变化小于 10%。由于 MPs 数量的变化和制备步骤中的损失，可以使用细胞骨架 MPs 蛋白强度对其进行归一化。我们的结果建立了一种可重复的 LC-MS/MS 程序，提供了一种简单而稳健的 MPs 制备方法，并为未来研究 MPs 在健康和疾病中的作用提供了 MPs 蛋白质组的基线。
