Franciosi Lorenza, Govorukhina Natalia, Ten Hacken Nick, Postma Dirkje, Bischoff Rainer
Department of Pharmacy, Analytical Biochemistry, University of Groningen, Groningen, The Netherlands.
Methods Mol Biol. 2011;790:17-28. doi: 10.1007/978-1-61779-319-6_2.
Epithelial lining fluid (ELF) forms a thin fluid layer that covers the mucosa of the alveoli, the small airways, and the large airways. Since it constitutes the first barrier between the lung and the outer world, it is an interesting target for proteomics studies that focus on lung disease. Bronchoscopic microprobe (BMP) sampling of ELF uses small probes with an absorptive tip that are introduced bronchoscopically. In contrast to other methods used so far for the collection of biofluids from the lung (e.g., bronchoalveolar lavage fluid, induced sputum), this technique has the advantage that ELF is not diluted and contains high concentrations of biomolecules. In addition, the investigated location in the tracheobronchial tree is well defined, and there is no contamination with oropharyngeal bacteria or saliva. Despite occasional blood contamination of the probes by scratching the mucosa of the airways, the proteomic analysis of microprobe-sampled ELF opens new possibilities for research in lung diseases. Our work focuses particularly on the induction and progression of cigarette smoke-induced Chronic Obstructive Pulmonary Disease (COPD). In this chapter, we describe the practical aspects of sampling ELF followed by a detailed description of proteomics analysis by LC-MS/MS after protein separation by SDS-PAGE and in-gel digestion. As an example, we apply this proteomic platform to the identification and quantification of proteins in ELF from COPD patients and healthy subjects.
上皮衬液（ELF）形成一层覆盖肺泡、小气道和大气道黏膜的薄液层。由于它构成了肺与外界之间的第一道屏障，因此是专注于肺部疾病的蛋白质组学研究的一个有趣目标。ELF的支气管镜微探针（BMP）采样使用带有吸收性尖端的小探针，通过支气管镜引入。与目前用于从肺部收集生物流体的其他方法（如支气管肺泡灌洗液、诱导痰）相比，该技术的优点是ELF不会被稀释，并且含有高浓度的生物分子。此外，气管支气管树中的研究位置明确，且不会被口咽细菌或唾液污染。尽管偶尔会因刮擦气道黏膜而使探针受到血液污染，但对微探针采样的ELF进行蛋白质组学分析为肺部疾病研究开辟了新的可能性。我们的工作特别关注香烟烟雾诱导的慢性阻塞性肺疾病（COPD）的诱导和进展。在本章中，我们描述了ELF采样的实际操作方面，随后详细描述了通过SDS-PAGE进行蛋白质分离和胶内消化后通过LC-MS/MS进行蛋白质组学分析的过程。作为一个例子，我们将这个蛋白质组学平台应用于COPD患者和健康受试者ELF中蛋白质的鉴定和定量。
