Proteomics

Dataset Information

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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]

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