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Abstract：
We present two separate label-free quantitative workflows based on different high-resolution mass spectrometers and LC setups, which are termed after the utilized instrument: Quad-Orbitrap (nano-LC) and Triple Quad-TOF (micro-LC) and their directed adaptation toward the analysis of human follicular fluid proteome. We identified about 1000 proteins in each distinct workflow using various sample preparation methods. With assistance of the Total Protein Approach, we were able to obtain absolute protein concentrations for each workflow. In a pilot study of twenty samples linked to diverse oocyte quality status from four donors, 455 and 215 proteins were quantified by the Quad-Orbitrap and Triple Quad-TOF workflows, respectively. The concentration values obtained from both workflows correlated to a significant degree. We found reasonable agreement of both workflows in protein fold changes between tested groups, resulting in unified lists of 20 and 22 proteins linked to oocyte maturity and blastocyst development, respectively. The Quad-Orbitrap workflow was best suited for an in-depth analysis without the need of extensive fractionation, especially of low abundant proteome, whereas the Triple Quad-TOF workflow allowed a more robust approach with a greater potential to increase in effectiveness with the growing number of analyzed samples after the initial effort of building a comprehensive spectral library.
摘要：
我们基于不同的高分辨率质谱仪和LC设置，提出了两个独立的无标签定量工作流程。在所使用的仪器之后称为：四轨道阱（nano-LC）和三四TOF（micro-LC）及其对人类卵泡液蛋白质组分析的定向适应。我们使用各种样品制备方法在每个不同的工作流程中鉴定了约1000种蛋白质。在总蛋白质方法的帮助下，我们能够获得每个工作流程的绝对蛋白质浓度.在一项对来自四个供体的与不同卵母细胞质量状态相关的20个样本的初步研究中，455和215蛋白质通过四轨道和三重四TOF工作流程进行了定量。分别。从两个工作流程获得的浓度值在显著程度上相关。我们发现测试组之间蛋白质折叠变化的两个工作流程的合理一致性，产生了与卵母细胞成熟和胚泡发育相关的20和22种蛋白质的统一列表，分别。Quad-Orbitrap工作流程最适合进行深入分析，而无需进行大量分馏，尤其是低丰度蛋白质组，而TripleQuad-TOF工作流程允许一种更稳健的方法,在构建全面的光谱库的最初努力之后,随着分析样本数量的增加,该方法具有更大的提高有效性的潜力。
