Dataset Information

Charting the interactome of PDE3A in human cells using an IBMX based chemical proteomics approach

ABSTRACT: We attempt to study the interactome of PDEs by synthesizing a broad spectrum PDE-capturing resin based on the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Chemical proteomics characterization of this resin in HeLa cell lysates led to the capture of several different PDEs. Combining the IBMX-resin with in-solution competition with other available, more selective PDE inhibitors, namely cilostamide and papaverine, allowed us to selectively probe the interactome of PDE3A in HeLa cells.

INSTRUMENT(S): TripleTOF 5600, LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Eleonora Corradini

LAB HEAD: Albert J.R. Heck

PROVIDER: PXD001781 | Pride | 2016-05-25

REPOSITORIES: Pride

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Publications

Charting the interactome of PDE3A in human cells using an IBMX based chemical proteomics approach.

Corradini Eleonora E Klaasse Gruson G Leurs Ulrike U Heck Albert J R AJ Martin Nathaniel I NI Scholten Arjen A

Molecular bioSystems 20151001 10

In the cell the second messenger cyclic nucleotides cAMP and cGMP mediate a wide variety of external signals. Both signaling molecules are degraded by the superfamily of phosphodiesterases (PDEs) consisting of more than 50 different isoforms. Several of these PDEs are implicated in disease processes inspiring the quest for and synthesis of selective PDE inhibitors, that unfortunately have led to very mixed successes in clinical trials. This may be partially caused by their pharmacological action ...[more]

PMID: 26205238

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Project description:The interaction between transcription factors and genomic DNA forms the basis for spatio-temporal control of gene expression. Therefore, these interactions and their impact on disease and cellular fate are extensively studied on a global level, mainly using techniques based on next-generation sequencing. These techniques, however, do not allow an unbiased study of proteins or entire protein complexes that bind to a certain DNA sequence. In recent years, DNA pull-downs followed by quantitative mass spectrometry were introduced to close this gap. Established protocols, however, are based on metabolic labeling techniques or require enormous amounts of cellular material, thus restricting the method to cell lines grown in culture. Furthermore, they require substantial amount of expertise, thus keeping this technique restricted to a limited number of laboratories. Here, we introduce a high-throughput compatible, LC-MS/MS based DNA pull-down that combines on-bead digestion with direct dimethyl labeling or label-free protein quantification. We demonstrate, that our method can efficiently identify transcription factors binding to their known consensus DNA motifs when using nuclear extracts from model cell lines. Subsequently, we apply the method to study DNA-protein interactions in primary foreskin fibroblasts and peripheral blood mononuclear cells (PBMCs) freshly isolated from human donors. We show that the same DNA sequence binds different sets of proteins in an established model cell line as opposed to PBMCs. This stresses the importance of selecting relevant cell extracts for any interaction in question. In-depth nuclear proteomes with absolute quantification of close to 7,000 proteins in K562 cells and PBMCs clearly link these differential interactions to differences in protein abundance. In conclusion, our approach, applicable to primary material and capable of profiling DNA-protein interactions in high-throughput, will likely prove itself as a useful screening platform and will provide invaluable functional data, for example through integration with large scale GWAS.

Dataset Information

Charting the interactome of PDE3A in human cells using an IBMX based chemical proteomics approach

Publications

Charting the interactome of PDE3A in human cells using an IBMX based chemical proteomics approach.

Similar Datasets

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