Dataset Information

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

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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]

PMID: 35134296

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Project description:Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. But, once the protein is already oxidized at least once, the spatial distribution of any consecutive oxidation no longer truly reflects initial protein solvent accessibility and residue reactivity, because the site preference of further oxidation may also be influenced by the changes caused by the prior oxidation. Thus, it would be interesting to obtain an assessment of the protein structure based on the FPOP data, by analyzing only singly oxidized protein populations. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is selection of a suitable method of ion isolation, excitation and fragmentation. Here we employ and compare collision-induced dissociation (CID), electron-transfer dissociation (ETD), and electron-capture dissociation (ECD) combined with multi continuous accumulation of selected ions (CASI). Singly oxidized subpopulation of FPOP labeled ubiquitin was used to optimize the method. The usefulness was then demonstrated further by using it to visualize structural changes induced by cofactor removal from holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in a good agreement, which indicates that monitoring singly FPOP oxidized ion population by top-down is a functional workflow for oxidative protein footprinting.

Dataset Information

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

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.

Similar Datasets

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