This readme file was generated on 2025-01-16 by Leah J. Bontreger ------------------- GENERAL INFORMATION ------------------- Title of Dataset: Data from: Intramolecular Histidine Crosslinks Formed via Copper-Catalyzed Oxidation of Histatin Peptides Author Contact Information Principal Investigator: Katherine J. Franz Institution: Duke University Email:katherine.franz@duke.edu ORCID: https://orcid.org/0000-0002-9015-0998 Associate or Co-investigator: Leah J. Bontreger Institution: Duke University Email: leah.bontreger@duke.edu ORCID: https://orcid.org/0009-0005-6877-6678 Alternate Contacts: Annastassia D. Gallo - Email: agallo@wooster.edu Jaewon Moon - Email: jaewon_moon@med.unc.edu Peter Silinski - Email: peter.silinski@duke.edu Eric Monson - Email: eric.monson@duke.edu Description of dataset: Histidine is a versatile amino acid with metal-binding, nucleophilic, and basic properties that endow many peptides and proteins with biological activity. However, histidine itself is susceptible to oxidative modifications via post-translational modifications, photooxidation, and metal-catalyzed oxidation. Despite multiple investigations into these different oxidation systems, the varied attributions and differential outcomes point to significant gaps in our understanding of the coordination requirements, spectral features, and reaction products that accompany Cu-catalyzed oxidation of histidine-containing peptides. Here, we use model peptides of Histatin-5, a salivary peptide with Cu-potentiated antifungal activity that relies on its histidine residues, to characterize the complex mixture resulting from reaction with Cu under physiologically relevant reducing and oxidizing conditions. Characterization via LC-MS, MS/MS, and NMR revealed adjacent histidine residues of the bis-His site were the main target of Cu-catalyzed oxidation, with predominant modifications being 2-oxo-His and His-His crosslinks. Doubly- and triply-oxygenated peptides, intramolecular His-His crosslinks, and multimers in the case of a shorter model peptide were also observed. The configuration of the bis-His motif may enable Cu reactivity not available in the other systems where His residues are not adjacent in sequence. These results expand the possibilities of oxidative modifications available to other proteins and peptides containing adjacent histidines. The NMR and MS data here characterized the various histidine modifications. The NMR data describes the protons, carbons, and the correlations between them, which provided rich structural detail for the peptide samples. The MS1 data showed the masses of ions from the native peptide and oxidized peptide products. These MS1 ions were sequenced by tandem MS to yield masses that correlated mass modifications to specific residues of the peptide. Data collected from 2022-02-25 to 2024-09-17 This project was funded in part by the US National Science Foundation, via CHE-1808710. -------------------- DATA & FILE OVERVIEW -------------------- NMR Data: Raw NMR folder: HHGY - folder for raw NMR data for peptide HHGY Info.txt - Text file detailing NMR sample preparation, collection, and processing HHGY_structure.mol - Mole file drawing of chemical structure of peptide HHGY 1H - folder for raw proton NMR experiment data including FID, processing parameters, and acquisition parameters 1H-13C HMBC - folder for raw proton - carbon heteronuclear multiple bond correlation NMR experiment data including FID, processing parameters, and acquisition parameters 13C - folder for raw carbon NMR experiment data including FID, processing parameters, and acquisition parameters oxHHGY - folder for raw NMR data for peptide mixture oxHHGY Info.txt - Text file detailing NMR sample preparation, collection, and processing oxHHGY_structure.mol - Mole file drawing of chemical structure of peptide oxHHGY 1H - folder for raw proton NMR experiment data including FID, processing parameters, and acquisition parameters 1H-13C HMBC - folder for raw proton - carbon heteronuclear multiple bond correlation NMR experiment data including FID, processing parameters, and acquisition parameters 13C - folder for raw carbon NMR experiment data including FID, processing parameters, and acquisition parameters Mass spectrometry data: MS1.csv - mass spectrometry ions and intensities of reaction mixture containing Native and oxidized Hist1-12 peptides, the following MS data files are collected by selecting ions in this MS1 file for tandem MS analysis 497.csv - tandem mass spectrometry of 497 ion, [M+3H]3+ of Native Hist1-12 503.csv - tandem mass spectrometry of 503 ion, [M+16+3H]3+ of Native Hist1-12, single oxygenation 508.csv - tandem mass spectrometry of 508 ion, [M+32+3H]3+ of Native Hist1-12, double oxygenation 513.csv - tandem mass spectrometry of 513 ion, [M+48+3H]3+ of Native Hist1-12, triple oxygenation 770.csv - tandem mass spectrometry of 770 ion, [M+48+2H]2+ of Native Hist1-12, triple oxygenation 497-xlink.csv - tandem mass spectrometry of 496 ion, [M-2+3H]3+ of Native Hist1-12, crosslink 746-xlink.csv - tandem mass spectrometry of 744 ion, [M-2+2H]2+ of Native Hist1-12, crosslink 513-xlink.csv - tandem mass spectrometry of 512 ion, [M+46+3H]3+ of Native Hist1-12, triple oxygenation and crosslink 770-xlink.csv - tandem mass spectrometry of 768 ion, [M+46+2H]2+ of Native Hist1-12, triple oxygenation and crosslink -------------------------- METHODOLOGICAL INFORMATION -------------------------- Description of methods used for collection/generation of data: NMR Sample Preparation, Collection, and Processing: Dried peptides were reconstituted with nanopure H2O and transferred to 3 mm tubes. A sealed capillary tube containing D2O with 1% trimethylsilane (TMS) was added to the sample. Spectra were collected on an Agilent/Varian DirectDrive2 800 MHz spectrometer at ambient temperature. The H2O signal was suppressed via a pre-saturation protocol. Spectra were acquired with Vnmr software and processed with Bruker TopSpin 4.1.1 software. Processing the data from Varian to TopSpin was accomplished according to this video: https://chembio.byu.edu/nmr-video-varian-topspin . Spectra were calibrated with TMS signal at 0 ppm. Mass Spectrometry Sample Preparation, Collection, and Analysis: For peptide sequencing analysis, a solution of 1 mM Hist1-12, 0.9 mM Cu(II), 5 mM ascorbate and 5 mM H2O2 at a total volume of 500 µL was reacted for 60 min and monitored by UV/vis spectroscopy. The sample was injected onto a reverse-phase HPLC prep column, and the fraction with 390 nm signal was collected, rotovapped, and lyophilized. The lyophilized solid was initially dissolved in water, then diluted into 90:10 water:acetonitrile with 0.1% formic acid. This sample was diluted again either ten times or 100 times in 90:10 water:acetonitrile. The samples were analyzed on a Thermo Orbitrap Exploris 480 instrument. Analysis was conducted by direct infusion using a syringe pump. For sequencing crosslinked masses, MS2 window was selected to be very narrow (1.5 ppm or less) in an effort to exclude ions from the native peptide. Theoretical masses were calculated with Microsoft Excel version 2412. Observed masses were recorded from most intense mass nearest calculated ion. For peptides sequenced at multiple charge states, observed masses were recorded using ions from both charge states. -------------------------- DATA-SPECIFIC INFORMATION -------------------------- Abbreviations: NMR - nuclear magnetic resonance MS - mass spectrometry HHGY - a peptide with sequence Histidine-Histidine-Glycine-Tyrosine oxHHGY - the mixture of peptide products resulting from HHGY copper catalyzed oxidation For Mass Spectrometry data: Variable/field list Mass = mass to charge ratio (m/z) of an observed ion Intensity = the height of the peak for the observed ion Value/attribute list Mass : given in units of Dalton Charge : an integer from 1-4, indicating how many positive charges the observed ion has Intensity : arbitrary units Missing data treatments Masses that were not observed are marked with '0' ------------------------- USE and ACCESS INFORMATION -------------------------- Data License: CC0 Other Rights Information: To cite the data: Franz, K. J., Bontreger, L. J., Gallo, A. D., Moon, J., Silinski, P., & Monson, E. (2025). Data from: Intramolecular Histidine Crosslinks Formed via Copper-Catalyzed Oxidation of Histatin Peptides. Duke Research Data Repository. https://doi.org/10.7924/r4nv9q11m.