Dataset Information

Determining DNA-protein binding affinities and specificities from crude lysates using a combined SILAC/TMT labeling strategy

ABSTRACT: Gene expression is driven by the binding of transcription factors to regulatory elements in the genome, such as enhancers and promoters. A powerful technique to study DNA-protein interactions is affinity purification followed by mass spectrometry. Classic affinity purifications coupled to quantitative mass spectrometry provide information about binding specificity. Binding of transcription factors to regulatory elements in vivo, however, also depends on binding affinity, so the strength of an interaction. To obtain this information, we recently developed a technique called PAQMAN that uses a series of DNA affinity purifications to quantify apparent binding affinities proteome-wide. Here, we expand our PAQMAN workflow to obtain information about binding specificity and affinity in a single experiment. To this end, we combine quantitation at the MS1 level with quantitation at the MS2 level, a strategy that is known as higher order multiplexing. This is, to our knowledge, the first time that higher order multiplexing is applied to affinity purification - mass spectrometry experiments. In the future, we anticipate that this new workflow will be a useful tool to investigate transcription factor biology.

INSTRUMENT(S): Orbitrap Fusion, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Cathrin Graewe

LAB HEAD: Michiel Vermeulen

PROVIDER: PXD041674 | Pride | 2023-10-08

REPOSITORIES: Pride

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Publications

Determining DNA-Protein Binding Affinities and Specificities from Crude Lysates Using a Combined SILAC/TMT Labeling Strategy.

Gräwe Cathrin C Hernandez-Quiles Miguel M Jansen Pascal W T C PWTC Brimmers Annika A Vermeulen Michiel M

Journal of proteome research 20230719 8

In recent years, quantitative mass spectrometry-based interaction proteomics technology has proven very useful in identifying specific DNA-protein interactions using single pull-downs from crude lysates. Here, we applied a SILAC/TMT-based higher-order multiplexing approach to develop an interaction proteomics workflow called Protein-nucleic acid Affinity and Specificity quantification by MAss spectrometry in Nuclear extracts or PASMAN. In PASMAN, DNA pull-downs using a concentration range of spe ...[more]

PMID: 37466164

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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

Determining DNA-protein binding affinities and specificities from crude lysates using a combined SILAC/TMT labeling strategy

Publications

Determining DNA-Protein Binding Affinities and Specificities from Crude Lysates Using a Combined SILAC/TMT Labeling Strategy.

Similar Datasets

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