Analysis of induced sputum supernatant is a minimally invasive approach to study the epithelial lining fluid and, thereby, provide insight into normal lung biology and the pathobiology of lung diseases. We present here a novel proteomics approach to sputum analysis developed within the U-BIOPRED (unbiased biomarkers predictive of respiratory disease outcomes) international project. We present practical and analytical techniques to optimize the detection of robust biomarkers in proteomic studies. The normal sputum proteome was derived using data-independent HDMS^E applied to 40 healthy nonsmoking participants, which provides an essential baseline from which to compare modulation of protein expression in respiratory diseases. The “core” sputum proteome (proteins detected in ≥40% of participants) was composed of 284 proteins, and the extended proteome (proteins detected in ≥3 participants) contained 1666 proteins. Quality control procedures were developed to optimize the accuracy and consistency of measurement of sputum proteins and analyze the distribution of sputum proteins in the healthy population. The analysis showed that quantitation of proteins by HDMS^E is influenced by several factors, with some proteins being measured in all participants’ samples and with low measurement variance between samples from the same patient. The measurement of some proteins is highly variable between repeat analyses, susceptible to sample processing effects, or difficult to accurately quantify by mass spectrometry. Other proteins show high interindividual variance. We also highlight that the sputum proteome of healthy individuals is related to sputum neutrophil levels, but not gender or allergic sensitization. We illustrate the importance of design and interpretation of disease biomarker studies considering such protein population and technical measurement variance.

中文翻译：

---

痰蛋白质组的大规模无标签定量映射。

诱导痰上清液的分析是一种微创方法，用于研究上皮衬里液，从而为正常的肺生物学和肺部疾病的病理学提供了见识。我们在这里介绍一种新的蛋白质组学方法，用于在U-BIOPRED（预测呼吸系统疾病结果的无偏性生物标志物）国际项目中开发的痰液分析方法。我们介绍了实用和分析技术，以优化蛋白质组学研究中强大的生物标志物的检测。正常痰液蛋白质组是使用与数据无关的HDMS ^E衍生的应用于40名健康的非吸烟参与者，该参与者提供了一个基本的基线，可以从中比较呼吸道疾病中蛋白质表达的调节。“核心”痰蛋白质组（在≥40％的参与者中检测到的蛋白质）由284种蛋白质组成，扩展的蛋白质组（在≥3名的参与者中检测到的蛋白质）包含1666个蛋白质。制定了质量控制程序，以优化痰蛋白测量的准确性和一致性，并分析健康人群中痰蛋白的分布。分析表明通过HDMS ^E对蛋白质进行定量它受多种因素的影响，在所有参与者的样品中都测量了某些蛋白质，同一患者的样品之间的测量方差很小。在重复分析之间，某些蛋白质的测量变化很大，容易受到样品处理的影响，或者难以通过质谱法准确定量。其他蛋白质表现出很高的个体差异。我们还强调，健康个体的痰液蛋白质组与痰液中性粒细胞水平有关，而与性别或过敏性敏化无关。我们阐述了考虑到这种蛋白质数量和技术测量差异的疾病生物标志物研究的设计和解释的重要性。