Haematoxylin and eosin (H&E)—which respectively stain nuclei blue and other cellular and stromal material pink—are routinely used for clinical diagnosis based on the identification of morphological features. A richer characterization can be achieved by laser capture microdissection coupled to mass spectrometry (LCM-MS), giving an unbiased assay of the proteins that make up the tissue. However, the process of fixing and H&E staining of tissues provides challenges with standard sample preparation methods for mass spectrometry, resulting in low protein yield. Here we describe a microproteomics technique to analyse H&E-stained, formalin-fixed paraffin-embedded (FFPE) tissues. Herein, we utilize heat extraction, physical disruption, and in column digestion for the analysis of H&E stained FFPE tissues. Micro-dissected morphologically normal human lung alveoli (0.082 mm3) and human lung blood vessels (0.094 mm3) from FFPE-fixed H&E-stained sections from Idiopathic Pulmonary Fibrosis (IPF) specimens (n = 3 IPF specimens) were then subject to a qualitative and then quantitative proteomics approach using BayesENproteomics. In addition, we tested the sensitivity of this method by processing and analysing a range of micro-dissected human lung blood vessel tissue volumes. This approach yields 1252 uniquely expressed proteins (at a protein identification threshold of 3 unique peptides) with 892 differentially expressed proteins between these regions. In accord with prior knowledge, our methodology approach confirms that human lung blood vessels are enriched with smoothelin, CNN1, ITGA7, MYH11, TAGLN, and PTGIS; whereas morphologically normal human lung alveoli are enriched with cytokeratin-7, -8, -18, -19, 14, and -17. In addition, we identify a total of 137 extracellular matrix (ECM) proteins and immunohistologically validate that laminin subunit beta-1 localizes to morphologically normal human lung alveoli and tenascin localizes to human lung blood vessels. Lastly, we show that this micro-proteomics technique can be applied to tissue volumes as low as 0.0125 mm3. Herein we show that our multistep sample preparation methodology of LCM-MS can identify distinct, characteristic proteomic compositions of anatomical features within complex fixed and stained tissues.

中文翻译：

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激光捕获显微切割耦合质谱 (LCM-MS) 用于对福尔马林固定和染色的人肺组织进行空间分辨分析。


苏木精和曙红 (H&E) 分别将细胞核染成蓝色，将其他细胞和基质材料染成粉红色，通常用于基于形态特征识别的临床诊断。通过激光捕获显微切割与质谱 (LCM-MS) 相结合，可以实现更丰富的表征，从而对构成组织的蛋白质进行公正的分析。然而，组织的固定和 H&E 染色过程对质谱分析的标准样品制备方法提出了挑战，导致蛋白质产量低。在这里，我们描述了一种微蛋白质组学技术来分析 H&E 染色、福尔马林固定石蜡包埋 (FFPE) 组织。在此，我们利用热提取、物理破碎和柱消化来分析 H&E 染色的 FFPE 组织。然后，对特发性肺纤维化 (IPF) 标本（n = 3 个 IPF 标本）的 FFPE 固定 H&E 染色切片进行显微解剖，形态正常的人肺泡 (0.082 mm3) 和人肺血管 (0.094 mm3) 进行定性分析然后使用 BayesENproteomics 进行定量蛋白质组学方法。此外，我们通过处理和分析一系列显微解剖的人体肺血管组织体积来测试该方法的灵敏度。这种方法产生了 1252 个独特表达的蛋白质（在 3 个独特肽的蛋白质识别阈值下），这些区域之间有 892 个差异表达的蛋白质。根据现有知识，我们的方法证实人类肺血管富含 smoothelin、CNN1、ITGA7、MYH11、TAGLN 和 PTGIS；而形态正常的人肺泡富含细胞角蛋白-7、-8、-18、-19、14 和-17。 此外，我们总共鉴定了 137 种细胞外基质 (ECM) 蛋白，并通过免疫组织学验证层粘连蛋白亚基 beta-1 定位于形态正常的人肺泡，腱蛋白定位于人肺血管。最后，我们证明这种微蛋白质组学技术可以应用于低至 0.0125 mm3 的组织体积。在此，我们表明，我们的 LCM-MS 多步样品制备方法可以识别复杂的固定和染色组织内解剖特征的独特的、特征性的蛋白质组组成。