Dataset Information

The Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Analysis.

ABSTRACT: 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.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Marek Polak

LAB HEAD: Marek Polak

PROVIDER: PXD044447 | Pride | 2024-01-26

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20220725_FOXO-IRE_000007.d.rar	Other
	20220725_FOXO-IRE_000008.d.rar	Other
	20220725_FOXO-IRE_000009.d.rar	Other
	20220725_FOXO_000007.d.rar	Other
	20220725_FOXO_000008.d.rar	Other

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Publications

Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Mass Spectrometry Analysis.

Polák Marek M Černý Jiří J Novák Petr P

Analytical chemistry 20240116 4

Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we ...[more]

PMID: 38226459

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