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

0585509 - MBÚ 2025 RIV US eng J - Článek v odborném periodiku
Polák, Marek - Černý, Jiří - Novák, Petr
Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Mass Spectrometry Analysis.
Analytical Chemistry. Roč. 96, č. 4 (2024), s. 1478-1487. ISSN 0003-2700. E-ISSN 1520-6882
Grant CEP: GA MŠMT(CZ) LX22NPO5107; GA ČR(CZ) GA22-27695S; GA MŠMT(CZ) LM2018127; GA MŠMT(CZ) LM2023055; GA MŠMT(CZ) ED1.1.00/02.0109
GRANT EU: European Commission(XE) 731077 - EU_FT-ICR_MS; European Commission(XE) 823839 - EPIC-XS
Výzkumná infrastruktura: CIISB II - 90127
Institucionální podpora: RVO:61388971 ; RVO:86652036
Klíčová slova: fast photochemical oxidation * amino-acid-residues * radiolytic modification * hydrogen-peroxide * structural probes * molecular-mass * cytochrome-c * protein * peptides * exchange
Obor OECD: Microbiology
Impakt faktor: 7.4, rok: 2022
Způsob publikování: Open access
https://pubs.acs.org/doi/10.1021/acs.analchem.3c03759

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 aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis. For this purpose, we prepared isotopically natural (IN) and depleted (ID) forms of the FOXO4 DNA binding domain (FOXO4-DBD) and studied the protein-DNA interaction interface with double-stranded DNA, the insulin response element (IRE), after exposing the complex to hydroxyl radicals. As shown by comparing tandem mass spectra of natural and depleted proteins, the ID form increased the signal-to-noise ratio of useful fragment ions, thereby enhancing the sequence coverage by more than 19%. This improvement in the detection of fragment ions enabled us to detect 22 more oxidized residues in the ID samples than in the IN sample. Moreover, less common modifications were detected in the ID sample, including the formation of ketones and lysine carbonylation. Given the higher quality of ID top-down MSMS data set, these results provide more detailed information on the complex formation between transcription factors and DNA-response elements. Therefore, our study highlights the benefits of isotopic depletion for quantitative top-down proteomics. Data are available via ProteomeXchange with the identifier PXD044447.
Trvalý link: https://hdl.handle.net/11104/0353192