Research Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic.
University of Edinburgh, Institute of Genetics and Molecular Medicine, Edinburgh, UK.
Biochim Biophys Acta Gen Subj. 2020 Dec;1864(12):129722. doi: 10.1016/j.bbagen.2020.129722. Epub 2020 Aug 29.
The identification of mutated proteins in human cancer cells-termed proteogenomics, requires several technologically independent research methodologies including DNA variant identification, RNA sequencing, and mass spectrometry. Any one of these methodologies are not optimized for identifying potential mutated proteins and any one output fails to cover completely a specific landscape.
An isogenic melanoma cell with a p53-null genotype was created by CRISPR/CAS9 system to determine how p53 gene inactivation affects mutant proteome expression. A mutant peptide reference database was developed by comparing two distinct DNA and RNA variant detection platforms using these isogenic cells. Chemically fractionated tryptic peptides from lysates were processed using a TripleTOF 5600+ mass spectrometer and their spectra were identified against this mutant reference database.
Approximately 190 mutated peptides were enriched in wt-p53 cells, 187 mutant peptides were enriched in p53-null cells, with an overlap of 147 mutated peptides. STRING analysis highlighted that the wt-p53 cell line was enriched for mutant protein pathways such as CDC5L and POLR1B, whilst the p53-null cell line was enriched for mutated proteins comprising EGF/YES, Ubiquitination, and RPL26/5 nodes.
Our study produces a well annotated p53-dependent and p53-independent mutant proteome of a common melanoma cell line model. Coupled to the application of an integrated DNA and RNA variant detection platform (CLCbio) and software for identification of proteins (ProteinPilot), this pipeline can be used to detect high confident mutant proteins in cells.
This pipeline forms a blueprint for identifying mutated proteins in diseased cell systems.
在人类癌细胞中鉴定突变蛋白——称为蛋白质基因组学，需要几种技术上独立的研究方法，包括 DNA 变异鉴定、RNA 测序和质谱分析。这些方法中的任何一种都没有针对潜在突变蛋白进行优化，任何一种方法的输出都不能完全覆盖特定的景观。
通过 CRISPR/CAS9 系统创建具有 p53 缺失基因型的同基因黑色素瘤细胞，以确定 p53 基因失活如何影响突变蛋白质组的表达。使用这些同基因细胞，通过比较两个不同的 DNA 和 RNA 变体检测平台，开发了一个突变肽参考数据库。从裂解物中化学分离的胰蛋白酶肽使用 TripleTOF 5600+质谱仪进行处理，并将其光谱与该突变参考数据库进行比较。
大约 190 个突变肽在 wt-p53 细胞中富集，187 个突变肽在 p53 缺失细胞中富集，重叠 147 个突变肽。STRING 分析强调，wt-p53 细胞系富集了 CDC5L 和 POLR1B 等突变蛋白途径，而 p53 缺失细胞系富集了包含 EGF/YES、泛素化和 RPL26/5 节点的突变蛋白。
我们的研究产生了一个注释良好的 p53 依赖和 p53 独立的常见黑色素瘤细胞系模型的突变蛋白质组。与集成的 DNA 和 RNA 变体检测平台（CLCbio）和用于鉴定蛋白质的软件（ProteinPilot）的应用相结合，该管道可用于检测细胞中的高置信突变蛋白。
该管道为鉴定疾病细胞系统中的突变蛋白提供了蓝图。
