Abstract:
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.
摘要:
将蛋白质基因组学与激光捕获显微切割(LCM)结合在癌症研究中提供了一种有针对性的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。这对于非常需要改善基于IO的药物的可预测性的免疫肿瘤学(IO)研究尤其重要。取决于对肿瘤空间关系的更好理解,血液供应,和免疫细胞相互作用,在它们相关的微环境中。LCM用于分离和获得不同的组织学细胞类型,这可以在复杂和异质实体瘤标本上常规进行。一旦细胞被捕获,核酸和蛋白质可以被提取用于深入的多模态分子谱分析测定。优化来自LCM捕获细胞的微小组织数量是具有挑战性的。在分离核酸之后,RNA-seq可用于基因表达,DNA测序可用于发现和分析可操作的突变。拷贝数变化,甲基化谱,等。然而,仍然需要针对小样本的高度敏感的蛋白质组学方法.该方案的重要部分是对微米级和/或纳米级组织切片的增强液相色谱质谱(LC-MS)分析。这是通过开发用于通过LCM获得的新鲜冷冻组织标本的LC-MS分析的银染色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。包括详细的凝胶内消化方法,经过调整和专门设计,以最大程度地提高限量型LCM样品的蛋白质组覆盖率,以更好地促进深入的分子谱分析。描述了从显微解剖的新鲜冷冻组织利用的蛋白质基因组方法。该方案还可以适用于具有有限核酸的其他类型的标本。蛋白质数量,和/或样品体积。