Background

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.

Methods

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.

Results

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.

Conclusion

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.

General significance

This pipeline forms a blueprint for identifying mutated proteins in diseased cell systems.

中文翻译：

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p53基因失活对人类黑素瘤细胞模型中突变蛋白质组表达的影响。

背景

在人类癌细胞中称为蛋白质组学的突变蛋白的鉴定，需要几种技术上独立的研究方法，包括DNA变体鉴定，RNA测序和质谱。这些方法中的任何一种都没有针对识别潜在突变蛋白进行优化，并且任何一种输出都无法完全涵盖特定的情况。

方法

CRISPR / CAS9系统创建了一个具有p53-null基因型的同基因黑素瘤细胞，以确定p53基因的失活如何影响突变蛋白质组的表达。通过使用这些等基因细胞比较两个不同的DNA和RNA变异检测平台，开发了一个突变肽参考数据库。使用TripleTOF 5600+质谱仪对来自裂解液的化学分级胰蛋白酶消化的肽进行处理，并根据该突变参考数据库鉴定其光谱。

结果

在wt-p53细胞中富集了大约190个突变肽，在p53无细胞中富集了187个突变肽，其中有147个突变肽重叠。STRING分析强调，wt-p53细胞株富含CDC5L和POLR1B等突变蛋白途径，而p53空细胞株富含EGF / YES，泛素化和RPL26 / 5节点的突变蛋白。

结论

我们的研究产生了常见的黑色素瘤细胞系模型的一个注释充分的p53依赖性和p53依赖性突变体蛋白质组。结合集成的DNA和RNA变异检测平台（CLCbio）和用于蛋白质鉴定的软件（ProteinPilot）的应用，该管道可用于检测细胞中的高可信度突变蛋白质。

一般意义

该管道形成了用于识别患病细胞系统中突变蛋白的蓝图。