PXD044447

PXD044447 is an original dataset announced via ProteomeXchange.

Title	The Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Analysis.
Description	Protein radical labeling offers an alternative analytical method for probing protein structure or protein interaction with other bio-molecules. Since the Fast Photochemical Oxidation of Proteins has already shown its essential role in studying biomolecular as-semblies and it was successfully adopted to characterize the interaction of transcription factor and its DNA response element, we initiated an experiment to investigate the benefits of isotopic depletion on analyzing the singly oxidized protein by Top-Down mass spectrometry. The complex of FOXO4 DNA-binding domain (FOXO4-DBD) and Insulin Response Element (IRE) was se-lected a model biological system. To overcome limitations of top-down technology which encounters predominantly with its spec-tra complexity, we prepared an isotopically depleted (ID) version of FOXO4-DBD alongside to the isotopically natural one (IN) to study the interaction. For the first time, depleted protein was used to quantify the extent of modification of covalently labelled protein. Comparing tandem mass spectra of natural and depleted proteins, increased signal-to-noise ratio gives arise to more frag-ment ions suitable for quantification and subsequently enhances the sequence coverage of 19 %. Such improvement in the frag-ment ions detection enables to detect additional 21 oxidized residues compered to non-depleted sample. Moreover, less common modifications are detected including formation of keto forms and lysine carbonylation. Moreover, the comparison of Top-Down depleted data and bottom-up results displays high consistency and complementarity of both techniques, shedding a light on tran-scription factor and DNA-response element complex formation. Thus, we believe that our study emphasizes the potential of iso-topic depletion for quantitative top-down proteomics.
HostingRepository	PRIDE
AnnounceDate	2024-01-26
AnnouncementXML	Submission_2024-01-26_06:03:06.963.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Marek Polak
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue
Instrument	timsTOF Pro

Revision	Datetime	Status	ChangeLog Entry
0	2023-08-08 03:52:29	ID requested
⏵ 1	2024-01-26 06:03:07	announced

10.1021/acs.analchem.3c03759;

Pol, á, k M, Č, ern, ý J, Nov, á, k P, Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Mass Spectrometry Analysis. Anal Chem, 96(4):1478-1487(2024) [pubmed]

submitter keyword: protein isotope depletion, IRE,Fast photochemical oxidation of proteins, mass spectrometry, Insulin response element, top-down, FOXO4, electron-capture dissociation, multiCASI.

Marek Polak
contact affiliation	Faculty of Science, Charles University, 12800 Prague, Czech Republic
contact email	marek.polak@biomed.cas.cz
lab head
Marek Polak
contact affiliation	Biocev - Institute of Microbiology of the Czech Academy of Science, Vestec, Czech Republic
contact email	marek.polak@biomed.cas.cz
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD044447
     1. Label: PRIDE project
     2. Name: The Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Analysis.