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首页> 外文期刊>Molecular & cellular proteomics: MCP >Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins
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Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins

机译：无稳定的无标记，定量分析循环等离子体微粒（MP）相关蛋白质

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Cells of the vascular system release spherical vesicles, called microparticles, in the size range of 0.1-1 m induced by a variety of stress factors resulting in variable concentrations between health and disease. Furthermore, microparticles have intercellular communication/signaling properties and interfere with inflammation and coagulation pathways. Today's most used analytical technology for microparticle characterization, flow cytometry, is lacking sensitivity and specificity, which might have led to the publication of contradicting results in the past. We propose the use of nano-liquid chromatography two-stage mass spectrometry as a nonbiased tool for quantitative MP proteome analysis. For this, we developed an improved microparticle isolation protocol and quantified the microparticle protein composition of twelve healthy volunteers with a label-free, data-dependent and independent proteomics approach on a quadrupole orbitrap instrument. Using aliquots of 250 l platelet-free plasma from one individual donor, we achieved excellent reproducibility with an interassay coefficient of variation of 2.7 +/- 1.7% (mean +/- 1 standard deviation) on individual peptide intensities across 27 acquisitions performed over a period of 3.5 months. We show that the microparticle proteome between twelve healthy volunteers were remarkably similar, and that it is clearly distinguishable from whole cell and platelet lysates. We propose the use of the proteome profile shown in this work as a quality criterion for microparticle purity in proteomics studies. Furthermore, one freeze thaw cycle damaged the microparticle integrity, articulated by a loss of cytoplasm proteins, encompassing a specific set of proteins involved in regulating dynamic structures of the cytoskeleton, and thrombin activation leading to MP clotting. On the other hand, plasma membrane protein composition was unaffected. Finally, we show that multiplexed data-independent acquisition can be used for relative quantification of target proteins using Skyline software. Mass spectrometry data are available via ProteomeXchange (identifier PXD003935) and panoramaweb.org (https://panoramaweb.org/labkey/N1OHMk.url).
机译：血管系统的细胞释放球形囊泡，称为微粒，尺寸范围为0.1-1m，由各种应力因子诱导，导致健​​康和疾病之间的可变浓度。此外，微粒具有细胞间通信/信号性质，并干扰炎症和凝血途径。今天的微粒表征最多使用的分析技术，流式细胞仪缺乏敏感性和特异性，这可能导致过去的出版物的矛盾。我们提出使用纳米液相色谱两级质谱法作为用于定量MP蛋白质组分析的非偏析工具。为此，我们开发了一种改进的微粒隔离方案，并在四肢侧玻璃仪器上用无标记，数据依赖性和独立的蛋白质组学方法量化了12个健康志愿者的微粒蛋白组成。使用来自一种单独供体的等分试样的血小板等离子体，我们实现了优异的再现性，其含量变异的含量为2.7 +/- 1.7％（平均+/-1.7％（平均+/- 1标准偏差），在27次采集上进行的个体肽强度3.5个月的时间。我们表明，十二份健康志愿者之间的微粒蛋白质组非常相似，并且从全细胞和血小板裂解物中明显区分。我们提出使用本工作中所示的蛋白质组曲线作为蛋白质组学研究中微粒纯度的质量标准。此外，一种冻解循环损坏了微粒完整性，通过丧失细胞质蛋白丧失铰接，包括参与调节细胞骨架的动态结构的特定蛋白质，以及导致MP凝血的凝血酶激活。另一方面，血浆膜蛋白组合物不受影响。最后，我们表明，使用Skyline软件可以使用多路复用的数据无关的采集来用于靶蛋白的相对定量。质谱数据可通过ProteomeXchange（标识符PXD003935）和PanoramaWeb.org（https://panoramaweb.org/labkey/n1ohmk.url）获得。

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