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

Abstract

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 confirm the findings and avoid a misinterpretation of the obtained data, caused by possible multiple radical oxidations leading to the protein surface alteration and oxidation of deeply buried amino acid residues, a top-down approach was employed for the first time in FPOP analysis. An isolation of singly oxidized ions enabled their gas-phase separation from multiply oxidized species followed by CID and ECD fragmentation. Application of both fragmentation techniques allowed generation of complementary fragment sets, out of which the regions shielded in the presence of DNA were deduced. The findings obtained by bottom-up and top-down approaches were highly consistent. Finally, FPOP results were compared with those of the HDX study of the FOXO4-DBD·DAF16 complex. No contradictions were found between the methods. Moreover, their combination provides complementary information related to the structure and dynamics of the protein–DNA complex. Data are available via ProteomeXchange with identifier PXD027624.

Rapid Online Oxidation of Proteins and Peptides via Electrospray-Accelerated Ozonation

“…This is dramatically different than the much higher yield and five oxidation events observed from the denatured protein (Figures B) . While top-down sequencing is necessary to assign the modification to a specific residue, four additional oxidation events are the expected result of increased Trp59 and Met65 accessibility. − …”

“…This species also displays an increase in labeling at 60% methanol indicating a reliance on the disruption of the hydrophobic core. To confidently assign the precise location of this oxidation event, future work coupling this labeling technique with an instrument platform capable of robust top-down sequencing is needed. − While other modes of denaturation still have to be examined, these results demonstrate that online ozone oxidation is sensitive to solution-driven protein denaturation similarly to other chemical labeling strategies. ,− …”

Top-Down Proteoform Analysis by 2D MS with Quadrupolar Detection

“…In mass spectrometry (MS)-based structural analysis of biomolecules, there are multiple methods available to probe three-dimensional structures: noncovalent labeling such as hydrogen–deuterium exchange, radical or oxidative foot-printing (for example, fast photochemical oxidation of proteins), amino acid- or base-selective probes, and chemical cross-linking. − The MS analysis of labeled biomolecules is performed by either a bottom-up or a top-down approach. − …”

“…New developments in ICR cells have enabled increased resolving power and SNR in FT-ICR MS, which have improved top-down approaches for protein footprinting techniques. , In mass spectrometers equipped with dynamically harmonized ICR cells, quadrupolar 2ω detection can be optimized with the appropriate electronics. By detecting ion signals at the 2ω harmonic, the resolving power can be doubled for a given transient length or the transient length can be halved for a given resolving power- .…”

Top-Down Detection of Oxidative Protein Footprinting by Collision-Induced Dissociation, Electron-Transfer Dissociation, and Electron-Capture Dissociation

“…That is why we are investigating the possibility of analyzing only proteins exposed to a single FPOP modification. In this respect, top-down MS offers an advantage over the bottom-up approach. − In the top-down workflow, the mass spectrum of intact protein ions is obtained, and ions of specific m / z values are further selected for fragmentation. When used for protein footprinting, it allows us to analyze only proteoforms with a specific degree of covalent modification. − In this work, we describe the use of top-down MS to study FPOP-labeled ubiquitin and utilization of FPOP to monitor conformational changes on myoglobin due to the removal of the heme group.…”