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Methods Enzymol. 2017;585:295-327. doi: 10.1016/bs.mie.2016.09.022. Epub 2016 Dec 3.

Combining Click Chemistry-Based Proteomics With Dox-Inducible Gene Expression.

Author information

1
Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; Cancer Early Detection, German Cancer Research Center (DKFZ), Heidelberg, Germany.
2
Functional Proteome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
3
Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; Cancer Early Detection, German Cancer Research Center (DKFZ), Heidelberg, Germany. Electronic address: juergen.kopitz@med.uni-heidelberg.de.

Abstract

Inactivating mutations in single genes can trigger, prevent, promote, or alleviate diseases. Identifying such disease-related genes is a main pillar of medical research. Since proteins play a crucial role in mediating these effects, their impact on the diseased cells' proteome including posttranslational modifications has to be elucidated for a detailed understanding of the role of these genes in the disease process. In complex disorders, like cancer, several genes contribute to the disease process, thereby hampering the assignment of a proteomic change to the corresponding causative gene. To enable comprehensive screening for the impact of inactivation of a gene, e.g., loss of a tumor suppressor in cancer, on the cellular proteome, we present a strategy based on combination of three technologies that is recombinase-mediated cassette exchange, click chemistry, and mass spectrometry. The methodology is exemplified by the analysis of the proteomic changes induced by the loss of a tumor suppressor gene in colorectal cancer cells. To demonstrate the applicability to screen for posttranslational modification changes, we also describe the analysis of protein glycosylation changes caused by the tumor suppressor inactivation. In principle, this strategy can be applied to analyze the effects of any gene of interest on protein expression as well as posttranslational modification by glycosylation. Moreover adaptation of the strategy to an appropriate cell culture model has the potential for application on a broad range of diseases where the disease-promoting mutations have been identified.

KEYWORDS:

Biotin labeling; Click chemistry; Genomic targeting; Glycome; Metabolic labeling; Proteome; Tumor suppressor

PMID:
28109436
DOI:
10.1016/bs.mie.2016.09.022
[Indexed for MEDLINE]

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