Schoenebeck Bodo, May Caroline, Güldner Christian, Respondek Gesine, Mollenhauer Brit, Hoeglinger Guenter, Meyer Helmut E, Marcus Katrin
Abteilung für Neuroanatomie und Molekulare Hirnforschung, Medizinische Fakultaet, Ruhr-Universitaet Bochum, 44801 Bochum, Germany.
Abteilung für Medizinische Proteomik/Bioanalytik, Medizinisches Proteom-Center, Ruhr-Universitaet Bochum, 44801 Bochum, Germany.
Biochim Biophys Acta. 2015 Jul;1854(7):741-5. doi: 10.1016/j.bbapap.2015.01.015. Epub 2015 Feb 10.
Nasal lavage fluid (NLF) becomes more and more important as a noninvasive patient sample serving as a new opportunity to discover new biomarkers in diverse human diseases comprising mainly respiratory disorders. This was supported by the observation that the protein profile of NLF differs from conventional samples of i.e. whole blood, hence being capable to complement or even expand the so far biomarker index. Since sample acquisition and processing are the most crucial steps for a profound and sensitive identification we present here a modified protocol of NLF generation and measurement. We show that mild washing steps for sample generation followed by column-mediated concentration and acetone precipitation are appropriate steps to minimize serum leakage by concomitantly highlighting proteins which represent typical components of NLF. This is shown by separation of proteins via 2D-PAGE followed by LC-MS/MS as well as Gel-LC-MS/MS measurements of cut and digested protein spots/bands.
For a better understanding of the molecular mechanisms underlying respiratory diseases NLF samples are favored sources for protein research. NLF acquisition and sample processing were impaired so far by the problem of blood serum leakage and high salt content. Here, we present a modified protocol of NLF generation leading to the display of typical inventory of NLF proteins combined with reduced salt and serum contaminants. By this, both assay reproducibility and the detection of up- or down-regulated proteins reliably can be discovered in the case of biomarker screenings in a disease versus control design. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.
鼻灌洗液（NLF）作为一种非侵入性患者样本，在发现包括主要呼吸系统疾病在内的多种人类疾病的新生物标志物方面成为越来越重要的新机会。NLF的蛋白质谱与全血等传统样本不同这一观察结果支持了这一点，因此它能够补充甚至扩展目前的生物标志物指数。由于样本采集和处理是进行深入和灵敏鉴定的最关键步骤，我们在此展示一种改良的NLF生成和测量方案。我们表明，样本生成时采用温和的洗涤步骤，随后进行柱介导浓缩和丙酮沉淀，这些步骤适合尽量减少血清泄漏，同时突出显示代表NLF典型成分的蛋白质。通过二维聚丙烯酰胺凝胶电泳（2D-PAGE）分离蛋白质，随后进行液相色谱-串联质谱（LC-MS/MS）以及对切割和消化后的蛋白质斑点/条带进行凝胶液相色谱-串联质谱测量，证明了这一点。
为了更好地理解呼吸系统疾病背后的分子机制，NLF样本是蛋白质研究的理想来源。到目前为止，血清泄漏和高盐含量问题一直影响着NLF的采集和样本处理。在此，我们展示一种改良的NLF生成方案，该方案能显示NLF蛋白质的典型成分，同时减少盐和血清污染物。通过这种方法，在疾病与对照设计的生物标志物筛查中，既能可靠地发现检测的可重复性，又能发现上调或下调蛋白质。本文是名为：神经蛋白质组学：在神经科学和神经病学中的应用的特刊的一部分。
