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.

Department of Biochemistry Faculty of Science Charles University 12843 Prague Czech Republic

Institute of Microbiology of the Czech Academy of Sciences 14220 Prague Czech Republic

Laboratory of Structural Bioinformatics of Proteins Institute of Biotechnology of the Czech Academy of Sciences 14220 Prague Czech Republic

Zobrazit více v PubMed

Polák M.; Yassaghi G.; Kavan D.; Filandr F.; Fiala J.; Kukačka Z.; Halada P.; Loginov D. S.; Novák P. 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. Anal. Chem. 2022, 94 (7), 3203–3210. 10.1021/acs.analchem.1c04746. PubMed DOI

Obsil T.; Obsilova V. Structural Basis for DNA Recognition by FOXO Proteins. Biochim. Biophys. Acta - Mol. Cell Res. 2011, 1813 (11), 1946–1953. 10.1016/j.bbamcr.2010.11.025. PubMed DOI

Vacha P.; Zuskova I.; Bumba L.; Herman P.; Vecer J.; Obsilova V.; Obsil T. Detailed Kinetic Analysis of the Interaction between the FOXO4–DNA-Binding Domain and DNA. Biophys. Chem. 2013, 184, 68–78. 10.1016/j.bpc.2013.09.002. PubMed DOI

Pandey P.; Hasnain S.; Ahmad S.. Protein-DNA Interactions. In Encyclopedia of Bioinformatics and Computational Biology; Elsevier, 2019; pp 142–154. 10.1016/B978-0-12-809633-8.20217-3. DOI

Lambert S. A.; Jolma A.; Campitelli L. F.; Das P. K.; Yin Y.; Albu M.; Chen X.; Taipale J.; Hughes T. R.; Weirauch M. T. The Human Transcription Factors. Cell 2018, 172 (4), 650–665. 10.1016/j.cell.2018.01.029. PubMed DOI

Hagenbuchner J.; Obsilova V.; Kaserer T.; Kaiser N.; Rass B.; Psenakova K.; Docekal V.; Alblova M.; Kohoutova K.; Schuster D.; Aneichyk T.; Vesely J.; Obexer P.; Obsil T.; Ausserlechner M. J. Modulating Foxo3 Transcriptional Activity by Small, Dbd-Binding Molecules. Elife 2019, 8, e48876.10.7554/eLife.48876. PubMed DOI PMC

Filandrová R.; Vališ K.; Černý J.; Chmelík J.; Slavata L.; Fiala J.; Rosůlek M.; Kavan D.; Man P.; Chum T.; Cebecauer M.; Fabris D.; Novák P. Motif Orientation Matters: Structural Characterization of TEAD1 Recognition of Genomic DNA. Structure 2021, 29 (4), 345–356.e8. 10.1016/j.str.2020.11.018. PubMed DOI

Slavata; Chmelík; Kavan; Filandrová; Fiala; Rosůlek; Mrázek; Kukačka; Vališ; Man; Miller; McIntyre; Fabris; Novák MS-Based Approaches Enable the Structural Characterization of Transcription Factor/DNA Response Element Complex. Biomolecules 2019, 9 (10), 535.10.3390/biom9100535. PubMed DOI PMC

Scalabrin M.; Dixit S. M.; Makshood M. M.; Krzemien C. E.; Fabris D. Bifunctional Cross-Linking Approaches for Mass Spectrometry-Based Investigation of Nucleic Acids and Protein-Nucleic Acid Assemblies. Methods 2018, 144, 64–78. 10.1016/j.ymeth.2018.05.001. PubMed DOI PMC

Sperry J. B.; Wilcox J. M.; Gross M. L. Strong Anion Exchange for Studying Protein-DNA Interactions by H/D Exchange Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19 (6), 887–890. 10.1016/j.jasms.2008.03.003. PubMed DOI PMC

Ma L.; Fitzgerald M. C. A New H/D Exchange- and Mass Spectrometry-Based Method for Thermodynamic Analysis of Protein-DNA Interactions. Chem. Biol. 2003, 10 (12), 1205–1213. 10.1016/j.chembiol.2003.11.017. PubMed DOI

Sperry J. B.; Shi X.; Rempel D. L.; Nishimura Y.; Akashi S.; Gross M. L. A Mass Spectrometric Approach to the Study of DNA-Binding Proteins: Interaction of Human TRF2 with Telomeric DNA. Biochemistry 2008, 47 (6), 1797–1807. 10.1021/bi702037p. PubMed DOI

Gau B. C.; Chen H.; Zhang Y.; Gross M. L. Sulfate Radical Anion as a New Reagent for Fast Photochemical Oxidation of Proteins. Anal. Chem. 2010, 82 (18), 7821–7827. 10.1021/ac101760y. PubMed DOI PMC

Chen J.; Cui W.; Giblin D.; Gross M. L. New Protein Footprinting: Fast Photochemical Iodination Combined with Top-Down and Bottom-Up Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2012, 23 (8), 1306–1318. 10.1007/s13361-012-0403-1. PubMed DOI PMC

Manzi L.; Barrow A. S.; Hopper J. T. S.; Kaminska R.; Kleanthous C.; Robinson C. V.; Moses J. E.; Oldham N. J. Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein. Angew. Chemie Int. Ed. 2017, 56 (47), 14873–14877. 10.1002/anie.201708254. PubMed DOI

Zhang M. M.; Rempel D. L.; Gross M. L. A Fast Photochemical Oxidation of Proteins (FPOP) Platform for Free-Radical Reactions: The Carbonate Radical Anion with Peptides and Proteins. Free Radic. Biol. Med. 2019, 131, 126–132. 10.1016/j.freeradbiomed.2018.11.031. PubMed DOI PMC

Cheng M.; Zhang B.; Cui W.; Gross M. L. Laser-Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass-Spectrometry-Based Protein Footprinting. Angew. Chemie - Int. Ed. 2017, 56 (45), 14007–14010. 10.1002/anie.201706697. PubMed DOI PMC

Fojtík L.; Fiala J.; Pompach P.; Chmelík J.; Matoušek V.; Beier P.; Kukačka Z.; Novák P. Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules. J. Am. Chem. Soc. 2021, 143 (49), 20670–20679. 10.1021/jacs.1c07771. PubMed DOI

Sharp J. S.; Becker J. M.; Hettich R. L. Protein Surface Mapping by Chemical Oxidation: Structural Analysis by Mass Spectrometry. Anal. Biochem. 2003, 313 (2), 216–225. 10.1016/S0003-2697(02)00612-7. PubMed DOI

Hambly D. M.; Gross M. L. Laser Flash Photolysis of Hydrogen Peroxide to Oxidize Protein Solvent-Accessible Residues on the Microsecond Timescale. J. Am. Soc. Mass Spectrom. 2005, 16 (12), 2057–2063. 10.1016/j.jasms.2005.09.008. PubMed DOI

Wang L.; Chance M. R. Protein Footprinting Comes of Age: Mass Spectrometry for Biophysical Structure Assessment. Mol. Cell. Proteomics 2017, 16 (5), 706–716. 10.1074/mcp.O116.064386. PubMed DOI PMC

Liu X. R.; Zhang M. M.; Gross M. L. Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications. Chem. Rev. 2020, 120 (10), 4355–4454. 10.1021/acs.chemrev.9b00815. PubMed DOI PMC

Xu G.; Chance M. R. Hydroxyl Radical-Mediated Modification of Proteins as Probes for Structural Proteomics. Chem. Rev. 2007, 107 (8), 3514–3543. 10.1021/cr0682047. PubMed DOI

Xu G.; Chance M. R. Radiolytic Modification of Acidic Amino Acid Residues in Peptides: Probes for Examining Protein–Protein Interactions. Anal. Chem. 2004, 76 (5), 1213–1221. 10.1021/ac035422g. PubMed DOI

Xu G.; Takamoto K.; Chance M. R. Radiolytic Modification of Basic Amino Acid Residues in Peptides: Probes for Examining Protein–Protein Interactions. Anal. Chem. 2003, 75 (24), 6995–7007. 10.1021/ac035104h. PubMed DOI

Xu G.; Chance M. R. Radiolytic Modification of Sulfur-Containing Amino Acid Residues in Model Peptides: Fundamental Studies for Protein Footprinting. Anal. Chem. 2005, 77 (8), 2437–2449. 10.1021/ac0484629. PubMed DOI

Charvátová O.; Foley B. L.; Bern M. W.; Sharp J. S.; Orlando R.; Woods R. J. Quantifying Protein Interface Footprinting by Hydroxyl Radical Oxidation and Molecular Dynamics Simulation: Application to Galectin-1. J. Am. Soc. Mass Spectrom. 2008, 19 (11), 1692–1705. 10.1016/j.jasms.2008.07.013. PubMed DOI PMC

Pan Y.; Stocks B. B.; Brown L.; Konermann L. Structural Characterization of an Integral Membrane Protein in Its Natural Lipid Environment by Oxidative Methionine Labeling and Mass Spectrometry. Anal. Chem. 2009, 81 (1), 28–35. 10.1021/ac8020449. PubMed DOI

Watkinson T. G.; Calabrese A. N.; Ault J. R.; Radford S. E.; Ashcroft A. E. FPOP-LC-MS/MS Suggests Differences in Interaction Sites of Amphipols and Detergents with Outer Membrane Proteins. J. Am. Soc. Mass Spectrom. 2017, 28 (1), 50–55. 10.1007/s13361-016-1421-1. PubMed DOI PMC

Lu Y.; Zhang H.; Niedzwiedzki D. M.; Jiang J.; Blankenship R. E.; Gross M. L. Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc. Anal. Chem. 2016, 88 (17), 8827–8834. 10.1021/acs.analchem.6b01945. PubMed DOI PMC

Gupta S.; Bavro V. N.; D’Mello R.; Tucker S. J.; Vénien-Bryan C.; Chance M. R. Conformational Changes during the Gating of a Potassium Channel Revealed by Structural Mass Spectrometry. Structure 2010, 18 (7), 839–846. 10.1016/j.str.2010.04.012. PubMed DOI PMC

Loginov D. S.; Fiala J.; Brechlin P.; Kruppa G.; Novak P. Hydroxyl Radical Footprinting Analysis of a Human Haptoglobin-Hemoglobin Complex. Biochim. Biophys. Acta - Proteins Proteomics 2022, 1870 (2), 14073510.1016/j.bbapap.2021.140735. PubMed DOI

Cornwell O.; Bond N. J.; Radford S. E.; Ashcroft A. E. Long-Range Conformational Changes in Monoclonal Antibodies Revealed Using FPOP-LC-MS/MS. Anal. Chem. 2019, 91 (23), 15163–15170. 10.1021/acs.analchem.9b03958. PubMed DOI

Cornwell O.; Radford S. E.; Ashcroft A. E.; Ault J. R. Comparing Hydrogen Deuterium Exchange and Fast Photochemical Oxidation of Proteins: A Structural Characterisation of Wild-Type and ΔN6 B2-Microglobulin. J. Am. Soc. Mass Spectrom. 2018, 29 (12), 2413–2426. 10.1007/s13361-018-2067-y. PubMed DOI PMC

Yassaghi G.; Kukačka Z.; Fiala J.; Kavan D.; Halada P.; Volný M.; Novák P. Top-Down Detection of Oxidative Protein Footprinting by Collision-Induced Dissociation, Electron-Transfer Dissociation, and Electron-Capture Dissociation. Anal. Chem. 2022, 94 (28), 9993–10002. 10.1021/acs.analchem.1c05476. PubMed DOI PMC

Tomášková N.; Novák P.; Kožár T.; Petrenčáková M.; Jancura D.; Yassaghi G.; Man P.; Sedlák E. Early Modification of Cytochrome c by Hydrogen Peroxide Triggers Its Fast Degradation. Int. J. Biol. Macromol. 2021, 174, 413–423. 10.1016/j.ijbiomac.2021.01.189. PubMed DOI

Donnelly D. P.; Rawlins C. M.; DeHart C. J.; Fornelli L.; Schachner L. F.; Lin Z.; Lippens J. L.; Aluri K. C.; Sarin R.; Chen B.; Lantz C.; Jung W.; Johnson K. R.; Koller A.; Wolff J. J.; Campuzano I. D. G.; Auclair J. R.; Ivanov A. R.; Whitelegge J. P.; Paša-Tolić L.; Chamot-Rooke J.; Danis P. O.; Smith L. M.; Tsybin Y. O.; Loo J. A.; Ge Y.; Kelleher N. L.; Agar J. N. Best Practices and Benchmarks for Intact Protein Analysis for Top-down Mass Spectrometry. Nat. Methods 2019, 16 (7), 587–594. 10.1038/s41592-019-0457-0. PubMed DOI PMC

Petrenčáková M.; Filandr F.; Hovan A.; Yassaghi G.; Man P.; Kožár T.; Schwer M. S.; Jancura D.; Plückthun A.; Novák P.; Miškovský P.; Bánó G.; Sedlák E. Photoinduced Damage of AsLOV2 Domain Is Accompanied by Increased Singlet Oxygen Production Due to Flavin Dissociation. Sci. Rep. 2020, 10 (1), 1–15. 10.1038/s41598-020-60861-2. PubMed DOI PMC

Kellersberger K. A.; Yu E.; Kruppa G. H.; Young M. M.; Fabris D. Top-Down Characterization of Nucleic Acids Modified by Structural Probes Using High-Resolution Tandem Mass Spectrometry and Automated Data Interpretation. Anal. Chem. 2004, 76 (9), 2438–2445. 10.1021/ac0355045. PubMed DOI

Valkenborg D.; Mertens I.; Lemière F.; Witters E.; Burzykowski T.. The Isotopic Distribution Conundrum. Mass Spectrometry Reviews; John Wiley & Sons, Ltd, January 1, 2012; pp 96–109. 10.1002/mas.20339. PubMed DOI

Compton P. D.; Zamdborg L.; Thomas P. M.; Kelleher N. L. On the Scalability and Requirements of Whole Protein Mass Spectrometry. Anal. Chem. 2011, 83 (17), 6868–6874. 10.1021/ac2010795. PubMed DOI PMC

Marshall A. G.; Senko M. W.; Li W.; Li M.; Dillon S.; Guan S.; Logan T. M. Protein Molecular Mass to 1 Da by 13 C, 15 N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119 (2), 433–434. 10.1021/ja9630046. DOI

Bou-Assaf G. M.; Chamoun J. E.; Emmett M. R.; Fajer P. G.; Marshall A. G. Advantages of Isotopic Depletion of Proteins for Hydrogen/Deuterium Exchange Experiments Monitored by Mass Spectrometry. Anal. Chem. 2010, 82 (8), 3293–3299. 10.1021/ac100079z. PubMed DOI PMC

Charlebois J. P.; Patrie S. M.; Kelleher N. L. Electron Capture Dissociation and 13C,15N Depletion for Deuterium Localization in Intact Proteins after Solution-Phase Exchange. Anal. Chem. 2003, 75 (13), 3263–3266. 10.1021/ac020690k. PubMed DOI

Zubarev R. A.; Demirev P. A. Isotope Depletion of Large Biomolecules: Implications for Molecular Mass Measurements. J. Am. Soc. Mass Spectrom. 1998, 9 (2), 149–156. 10.1016/S1044-0305(97)00232-8. DOI

Gallagher K. J.; Palasser M.; Hughes S.; Mackay C. L.; Kilgour D. P. A.; Clarke D. J. Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins. J. Am. Soc. Mass Spectrom. 2020, 31 (3), 700–710. 10.1021/jasms.9b00119. PubMed DOI

Popovic Z.; Anderson L. C.; Zhang X.; Butcher D. S.; Blakney G. T.; Zubarev R. A.; Marshall A. G. Analysis of Isotopically Depleted Proteins Derived from Escherichia Coli and Caenorhabditis Elegans Cell Lines by Liquid Chromatography 21 T Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2023, 34, 137–144. 10.1021/jasms.2c00242. PubMed DOI

Loginov D. S.; Fiala J.; Chmelik J.; Brechlin P.; Kruppa G.; Novak P. Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on TimsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis. ACS Omega 2021, 6 (15), 10352–10361. 10.1021/acsomega.1c00732. PubMed DOI PMC

Li K. S.; Shi L.; Gross M. L. Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization. Acc. Chem. Res. 2018, 51 (3), 736–744. 10.1021/acs.accounts.7b00593. PubMed DOI PMC

Perez-Riverol Y.; Bai J.; Bandla C.; García-Seisdedos D.; Hewapathirana S.; Kamatchinathan S.; Kundu D. J.; Prakash A.; Frericks-Zipper A.; Eisenacher M.; Walzer M.; Wang S.; Brazma A.; Vizcaíno J. A. The PRIDE Database Resources in 2022: A Hub for Mass Spectrometry-Based Proteomics Evidences. Nucleic Acids Res. 2022, 50 (D1), D543–D552. 10.1093/nar/gkab1038. PubMed DOI PMC

Boura E.; Rezabkova L.; Brynda J.; Obsilova V.; Obsil T. Structure of the Human FOXO4-DBD–DNA Complex at 1.9 Å Resolution Reveals New Details of FOXO Binding to the DNA. Acta Crystallogr. Sect. D Biol. Crystallogr. 2010, 66 (12), 1351–1357. 10.1107/S0907444910042228. PubMed DOI

Flores S. C.; Altman R. B. Turning Limited Experimental Information into 3D Models of RNA. RNA 2010, 16 (9), 1769–1778. 10.1261/rna.2112110. PubMed DOI PMC

Flores S. C.; Bernauer J.; Shin S.; Zhou R.; Huang X. Multiscale Modeling of Macromolecular Biosystems. Brief. Bioinform. 2012, 13 (4), 395–405. 10.1093/bib/bbr077. PubMed DOI

Černý J.; Božíková P.; Svoboda J.; Schneider B. A Unified Dinucleotide Alphabet Describing Both RNA and DNA Structures. Nucleic Acids Res. 2020, 48 (11), 6367–6381. 10.1093/nar/gkaa383. PubMed DOI PMC

Černý J.; Božíková P.; Malý M.; Tykač M.; Biedermannová L.; Schneider B. Structural Alphabets for Conformational Analysis of Nucleic Acids Available at Dnatco.Datmos.Org. Acta Crystallogr. Sect. D Struct. Biol. 2020, 76 (9), 805–813. 10.1107/S2059798320009389. PubMed DOI PMC

Abraham M. J.; Murtola T.; Schulz R.; Páll S.; Smith J. C.; Hess B.; Lindahl E. GROMACS: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers. SoftwareX 2015, 1–2, 19–25. 10.1016/j.softx.2015.06.001. DOI

Maier J. A.; Martinez C.; Kasavajhala K.; Wickstrom L.; Hauser K. E.; Simmerling C. Ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from Ff99SB. J. Chem. Theory Comput. 2015, 11 (8), 3696–3713. 10.1021/acs.jctc.5b00255. PubMed DOI PMC

Liebl K.; Zacharias M. Tumuc1: A New Accurate DNA Force Field Consistent with High-Level Quantum Chemistry. J. Chem. Theory Comput. 2021, 17 (11), 7096–7105. 10.1021/acs.jctc.1c00682. PubMed DOI

Xu G.; Chance M. R. Radiolytic Modification and Reactivity of Amino Acid Residues Serving as Structural Probes for Protein Footprinting. Anal. Chem. 2005, 77 (14), 4549–4555. 10.1021/ac050299+. PubMed DOI

Niu B.; Gross M. L.. MS-Based Hydroxyl Radical Footprinting: Methodology and Application of Fast Photochemical Oxidation of Proteins (FPOP). In Mass Spectrometry-Based Chemical Proteomics; Wiley, 2019; pp 363–416. 10.1002/9781118970195.ch15. DOI

Yin V.; Mian S. H.; Konermann L. Lysine Carbonylation Is a Previously Unrecognized Contributor to Peroxidase Activation of Cytochrome c by Chloramine-T. Chem. Sci. 2019, 10 (8), 2349–2359. 10.1039/C8SC03624A. PubMed DOI PMC

Liu X. R.; Zhang M. M.; Gross M. L. Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications. Chemical Reviews 2020, 4355–4454. 10.1021/acs.chemrev.9b00815. PubMed DOI PMC

Boura E.; Silhan J.; Herman P.; Vecer J.; Sulc M.; Teisinger J.; Obsilova V.; Obsil T. Both the N-Terminal Loop and Wing W2 of the Forkhead Domain of Transcription Factor Foxo4 Are Important for DNA Binding. J. Biol. Chem. 2007, 282 (11), 8265–8275. 10.1074/jbc.M605682200. PubMed DOI

Obsilova V.; Vecer J.; Herman P.; Pabianova A.; Sulc M.; Teisinger J.; Boura E.; Obsil T. 14–3-3 Protein Interacts with Nuclear Localization Sequence of Forkhead Transcription Factor FoxO4. Biochemistry 2005, 44 (34), 11608–11617. 10.1021/bi050618r. PubMed DOI

James V. K.; Sanders J. D.; Aizikov K.; Fort K. L.; Grinfeld D.; Makarov A.; Brodbelt J. S. Advancing Orbitrap Measurements of Collision Cross Sections to Multiple Species for Broad Applications. Anal. Chem. 2022, 94 (45), 15613–15620. 10.1021/acs.analchem.2c02146. PubMed DOI

Data-Independent Acquisition Represents a Promising Alternative for Fast Photochemical Oxidation of Proteins (FPOP) Samples Analysis