Fast Photochemical Oxidation of Proteins (FPOP) is a promising technique for studying protein structure and dynamics. The quality of insight provided by FPOP depends on the reliability of the determination of the modification site. This study investigates the performance of two search engines, Mascot and PEAKS, for the data processing of FPOP analyses. Comparison of Mascot and PEAKS of the hemoglobin--haptoglobin Bruker timsTOF data set (PXD021621) revealed greater consistency in the Mascot identification of modified peptides, with around 26% of the IDs being mutual for all three replicates, compared to approximately 22% for PEAKS. The intersection between Mascot and PEAKS results revealed a limited number (31%) of shared modified peptides. Principal Component Analysis (PCA) using the peptide-spectrum match (PSM) score, site probability, and peptide intensity was applied to evaluate the results, and the analyses revealed distinct clusters of modified peptides. Mascot showed the ability to assess confident site determination, even with lower PSM scores. However, high PSM scores from PEAKS did not guarantee a reliable determination of the modification site. Fragmentation coverage of the modification position played a crucial role in Mascot assignments, while the AScore localizations from PEAKS often become ambiguous because the software employs MS/MS merging.

Department of Biochemistry Faculty of Science Charles University 12820 Prague Czech Republic

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

Zobrazit více v PubMed

Pan J.; Zhang S.; Borchers C. H. Comparative Higher-Order Structure Analysis of Antibody Biosimilars Using Combined Bottom-up and Top-down Hydrogen-Deuterium Exchange Mass Spectrometry. Biochim. Biophys. Acta 2016, 1864 (12), 1801–1808. 10.1016/j.bbapap.2016.08.013. PubMed DOI

Liu F.; Rijkers D. T. S.; Post H.; Heck A. J. R. Proteome-Wide Profiling of Protein Assemblies by Cross-Linking Mass Spectrometry. Nat. Methods 2015, 12, 1179–1184. 10.1038/nmeth.3603. PubMed DOI

Leney A. C.; Rafie K.; Van Aalten D. M. F.; Heck A. J. R. Direct Monitoring of Protein O-GlcNAcylation by High-Resolution Native Mass Spectrometry. ACS Chem. Biol. 2017, 12 (8), 2078–2084. 10.1021/acschembio.7b00371. PubMed DOI PMC

Zhang Z.; Smith D. L. Determination of Amide Hydrogen Exchange by Mass Spectrometry: A New Tool for Protein Structure Elucidation. Protein Sci. 1993, 2 (4), 522–531. 10.1002/pro.5560020404. PubMed DOI PMC

Brown K. A.; Wilson D. J. Bottom-up Hydrogen Deuterium Exchange Mass Spectrometry: Data Analysis and Interpretation. Analyst 2017, 142 (16), 2874–2886. 10.1039/C7AN00662D. PubMed DOI

Lau A. M.; Claesen J.; Hansen K.; Politis A. Deuteros 2.0: Peptide-Level Significance Testing of Data from Hydrogen Deuterium Exchange Mass Spectrometry. Bioinformatics 2021, 37 (2), 270–272. 10.1093/bioinformatics/btaa677. PubMed DOI PMC

Narang D.; Lento C.; Wilson D. J. HDX-MS: An Analytical Tool to Capture Protein Motion in Action. Biomedicines 2020, 8 (7), 224.10.3390/biomedicines8070224. PubMed DOI PMC

Vávra J.; Sergunin A.; Stráňava M.; Kádek A.; Shimizu T.; Man P.; Martínková M. Hydrogen/Deuterium Exchange Mass Spectrometry of Heme-Based Oxygen Sensor Proteins. Methods Mol. Biol. Clifton NJ. 2023, 2648, 99–122. 10.1007/978-1-0716-3080-8_8. PubMed DOI

Cheng M.; Zhang B.; Cui W.; Gross M. L. Laser-Initiated Radical Trifluoromethylation of Peptides and Proteins and Its Application to Mass Spectrometry-Based Protein Footprinting HHS Public Access. Angew. Chem., Int. Ed. Engl. 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

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

Smith R. A. G.; Knowles J. R. Letter: Aryldiazirines. Potential Reagents for Photolabeling of Biological Receptor Sites. J. Am. Chem. Soc. 1973, 95 (15), 5072–5073. 10.1021/ja00796a062. 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

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

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

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

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, 8827.10.1021/acs.analchem.6b01945. PubMed DOI PMC

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

Liu X. R.; Rempel D. L.; Gross M. L. Protein Higher-Order-Structure Determination by Fast Photochemical Oxidation of Proteins and Mass Spectrometry Analysis. Nat. Protoc. 2020, 15 (12), 3942–3970. 10.1038/s41596-020-0396-3. PubMed DOI PMC

Rinas A.; Espino J. A.; Jones L. M. An Efficient Quantitation Strategy for Hydroxyl Radical-Mediated Protein Footprinting Using Proteome Discoverer. Anal. Bioanal. Chem. 2016, 408 (11), 3021–3031. 10.1007/s00216-016-9369-3. PubMed DOI

Chea E. E.; Prakash A.; Jones L. M. The Utilization of the Search Engine, Bolt, to Decrease Search Time and Increase Peptide Identifications in Hydroxyl Radical Protein Footprinting-Based Workflows. Proteomics 2021, 21 (21–22), 2000295.10.1002/pmic.202000295. PubMed DOI

Ziemianowicz D. S.; Sarpe V.; Schriemer D. C. Quantitative Analysis of Protein Covalent Labeling Mass Spectrometry Data in the Mass Spec Studio. Anal. Chem. 2019, 91 (13), 8492–8499. 10.1021/acs.analchem.9b01625. PubMed DOI

Bellamy-Carter J.; Oldham N. J. PepFoot: A Software Package for Semiautomated Processing of Protein Footprinting Data. J. Proteome Res. 2019, 18 (7), 2925–2930. 10.1021/acs.jproteome.9b00238. PubMed DOI

Kong A. T.; Leprevost F. V.; Avtonomov D. M.; Mellacheruvu D.; Nesvizhskii A. I. MSFragger: Ultrafast and Comprehensive Peptide Identification in Shotgun Proteomics. Nat. Methods 2017, 14 (5), 513–520. 10.1038/nmeth.4256. PubMed DOI PMC

Eng J. K.; Jahan T. A.; Hoopmann M. R. Comet: An Open-Source MS/MS Sequence Database Search Tool. PROTEOMICS 2013, 13 (1), 22–24. 10.1002/pmic.201200439. PubMed DOI

Kim S.; Pevzner P. A. MS-GF+ Makes Progress towards a Universal Database Search Tool for Proteomics. Nat. Commun. 2014, 5, 5277.10.1038/ncomms6277. PubMed DOI PMC

Yuan Z. F.; Lin S.; Molden R. C.; Garcia B. A. Evaluation of Proteomic Search Engines for the Analysis of Histone Modifications. J. Proteome Res. 2014, 13 (10), 4470–4478. 10.1021/pr5008015. PubMed DOI PMC

Perkins D. N.; Pappin D. J. C.; Creasy D. M.; Cottrell J. S. Probability-Based Protein Identification by Searching Sequence Databases Using Mass Spectrometry Data. ELECTROPHORESIS 1999, 20 (18), 3551–3567. 10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2. PubMed DOI

Zhang J.; Xin L.; Shan B.; Chen W.; Xie M.; Yuen D.; Zhang W.; Zhang Z.; Lajoie G. A.; Ma B. PEAKS DB: De Novo Sequencing Assisted Database Search for Sensitive and Accurate Peptide Identification. Mol. Cell. Proteomics 2012, 11 (4), M111.010587.10.1074/mcp.M111.010587. PubMed DOI PMC

Savitski M. M.; Lemeer S.; Boesche M.; Lang M.; Mathieson T.; Bantscheff M.; Kuster B. Confident Phosphorylation Site Localization Using the Mascot Delta Score. Mol. Cell. Proteomics 2011, 10 (2), S1–S12. 10.1074/mcp.M110.003830. PubMed DOI PMC

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

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), 140735.10.1016/j.bbapap.2021.140735. PubMed DOI

Deutsch E. W.; Bandeira N.; Perez-Riverol Y.; Sharma V.; Carver J. J.; Mendoza L.; Kundu D. J.; Wang S.; Bandla C.; Kamatchinathan S.; Hewapathirana S.; Pullman B. S.; Wertz J.; Sun Z.; Kawano S.; Okuda S.; Watanabe Y.; MacLean B.; MacCoss M. J.; Zhu Y.; Ishihama Y.; Vizcaíno J. A. The ProteomeXchange Consortium at 10 Years: 2023 Update. Nucleic Acids Res. 2023, 51 (D1), D1539–D1548. 10.1093/nar/gkac1040. PubMed DOI PMC

Pompach P.; Brnakova Z.; Sanda M.; Wu J.; Edwards N.; Goldman R. Site-Specific Glycoforms of Haptoglobin in Liver Cirrhosis and Hepatocellular Carcinoma. Mol. Cell. Proteomics 2013, 12 (5), 1281–1293. 10.1074/mcp.M112.023259. PubMed DOI PMC

Tretyakov K.Area-Weighted Venn-Diagrams for Python/Matplotlib; https://github.com/konstantint/matplotlib-venn (accessed 2023-03-03).

R Core Team . R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing, 2022; https://www.R-project.org/.

Lê S.; Josse J.; Husson F. FactoMineR: An R Package for Multivariate Analysis. J. Stat. Softw. 2008, 25 (1), 1–18. 10.18637/jss.v025.i01. DOI

Kassambara A.; Mundt F.. Factoextra: Extract and Visualize the Results of Multivariate Data Analyses; 2020; https://CRAN.R-project.org/package=factoextra (accessed 2023-03-03).

Wei T.; Simko V.. R Package “Corrplot”: Visualization of a Correlation Matrix; 2021; https://github.com/taiyun/corrplot (accessed 2023-03-03).

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 β 2-Microglobulin. J. Am. Soc. Mass Spectrom. 2018, 29, 2413–2426. 10.1007/s13361-018-2067-y. PubMed DOI PMC

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