Increasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Methods allowing the isolation of histones from patient samples followed by the accurate and comprehensive quantification of their PTMs by mass spectrometry (MS) have been developed. However, the applicability of these methods is limited by the requirement for substantial amounts of material. To address this issue, in this study we streamlined the protein extraction procedure from low-amount clinical samples and tested and implemented different in-gel digestion strategies, obtaining a protocol that allows the MS-based analysis of the most common histone PTMs from laser microdissected tissue areas containing as low as 1000 cells, an amount approximately 500 times lower than what is required by available methods. We then applied this protocol to breast cancer patient laser microdissected tissues in two proof-of-concept experiments, identifying differences in histone marks in heterogeneous regions selected by either morphological evaluation or MALDI MS imaging. These results demonstrate that analyzing histone PTMs from very small tissue areas and detecting differences from adjacent tumor regions is technically feasible. Our method opens the way for spatial epi-proteomics, namely the investigation of epigenetic features in the context of tissue and tumor heterogeneity, which will be instrumental for the identification of novel epigenetic biomarkers and aberrant epigenetic mechanisms.

中文翻译：

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通过低丰度临床样本的组蛋白翻译后修饰分析实现空间表观蛋白质组学

越来越多的证据将表观遗传机制与包括癌症在内的不同疾病联系起来，促使人们在过去 15 年中对临床样本中的组蛋白翻译后修饰 (PTM) 进行研究。已经开发出从患者样本中分离组蛋白，然后通过质谱 (MS) 对其 PTM 进行准确、全面量化的方法。然而，这些方法的适用性受到对大量材料的要求的限制。为了解决这个问题，在本研究中，我们简化了从少量临床样品中提取蛋白质的程序，并测试和实施了不同的凝胶内消化策略，获得了一个协议，该协议允许对激光显微切割中最常见的组蛋白 PTM 进行基于 MS 的分析组织区域含有低至 1000 个细胞，数量比现有方法所需的数量低约 500 倍。然后，我们在两个概念验证实验中将该方案应用于乳腺癌患者激光显微切割组织，识别通过形态学评估或 MALDI MS 成像选择的异质区域中组蛋白标记的差异。这些结果表明，分析非常小的组织区域的组蛋白 PTM 并检测邻近肿瘤区域的差异在技术上是可行的。我们的方法为空间表观蛋白质组学开辟了道路，即在组织和肿瘤异质性背景下研究表观遗传特征，这将有助于识别新型表观遗传生物标志物和异常表观遗传机制。