Proteomics

Dataset Information

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Structural aspects of FOXO4 binding to DAF16 investigated with FPOP using both bottom-up and top-down MS.


ABSTRACT: A combination of covalent labelling techniques and mass spectrometry (MS) is currently a progressive approach for the de-riving insights relating to the mapping of protein surfaces or protein-ligand interactions. In this study, we mapped an interac-tion interface between DNA binding domain (DBD) of FOXO4 protein and DNA binding element (DAF16) using the Fast Photochemical Oxidation of Protein (FPOP). Residues involved in protein – DNA interaction were identified using bottom-up approach. To avoid a misinterpretation of obtained data, caused by possible multiple radical oxidation leading to the alter-ing a protein surface and oxidation of deep-buried amino acid residues, a top-down approach was employed for the first time in FPOP analysis. An isolation of singly-oxidized ions allowed their gas-phase separation from multiply oxidized species followed by CID and ECD fragmentation. Application of both fragmentation techniques allowed to obtained complemen-tary fragment sets, out of which the regions shielded in the presence of DNA were deduced. The results obtained by bottom-up and top-down approaches were in a high consistency. Lastly, FPOP results were compared with HDX study of FOXO4-DBD•DAF16 complex. No contradictions were found between both methods. Moreover, their combination provide com-plementary information related to the structure and dynamics of the protein-DNA complex.

INSTRUMENT(S): solariX

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Marek Polak  

LAB HEAD: Petr Novak

PROVIDER: PXD027624 | Pride | 2022-02-14

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
Bottom-up_LCMSMS.zip Other
Bottom-up_raw_LCMS.zip Other
FOXO4.fasta Fasta
LysC_search.csv Csv
Ms2Links_files.zip Other
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Publications

Utilization of Fast Photochemical Oxidation of Proteins and Both Bottom-up and Top-down Mass Spectrometry for Structural Characterization of a Transcription Factor-dsDNA Complex.

Polák Marek M   Yassaghi Ghazaleh G   Kavan Daniel D   Filandr František F   Fiala Jan J   Kukačka Zdeněk Z   Halada Petr P   Loginov Dmitry S DS   Novák Petr P  

Analytical chemistry 20220208 7


A combination of covalent labeling techniques and mass spectrometry (MS) is currently a progressive approach for deriving insights related to the mapping of protein surfaces or protein-ligand interactions. In this study, we mapped an interaction interface between the DNA binding domain (DBD) of FOXO4 protein and the DNA binding element (DAF16) using fast photochemical oxidation of proteins (FPOP). Residues involved in protein-DNA interaction were identified using the bottom-up approach. To confi  ...[more]

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