Winthrop P Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
Department of Biology, Loyola University Maryland, Baltimore, MD, USA.
Methods Mol Biol. 2024;2823:55-75. doi: 10.1007/978-1-0716-3922-1_5.
Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.
在癌症研究中，将蛋白质组学与激光捕获显微切割（LCM）相结合，提供了一种靶向方法来探索肿瘤细胞与不同微环境成分之间的复杂相互作用。这在免疫肿瘤学（IO）研究中尤为重要，因为迫切需要提高 IO 药物的可预测性，而这依赖于更好地理解涉及肿瘤、血液供应和免疫细胞相互作用的空间关系，以及它们在相关微环境中的相互作用。LCM 用于分离和获得不同的组织学细胞类型，通常可在复杂和异质的实体肿瘤标本上进行。一旦捕获了细胞，就可以提取核酸和蛋白质，用于深入的多模态分子分析。从 LCM 捕获的细胞中优化微量组织量是具有挑战性的。在分离核酸之后，可以进行 RNA-seq 以进行基因表达，进行 DNA 测序以发现和分析可操作的突变、拷贝数变异、甲基化谱等。然而，仍然需要针对小样本量的高度敏感的蛋白质组学方法。该方案的一个重要部分是针对微尺度和/或纳米尺度组织切片的增强型液相色谱-质谱（LC-MS）分析。这是通过开发用于 LC-MS 分析 LCM 获得的新鲜冷冻组织标本的银染一维十二烷基硫酸钠聚丙烯酰胺凝胶电泳（1D-SDS-PAGE）方法实现的。包括详细的胶内消化方法，该方法经过调整和专门设计，可从有限量的 LCM 样本中最大限度地提高蛋白质组覆盖度，以更好地促进深入的分子分析。介绍了一种利用从微切割新鲜冷冻组织中获得的蛋白质组学方法。该方案也可能适用于具有有限核酸、蛋白质量和/或样品体积的其他类型的标本。
