Tyrosine halogenation converts α-defensins into potent immunomodulators
Data files
Nov 06, 2026 version files 34.26 MB
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Data_S1.xlsx
34.21 MB
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README.md
55.70 KB
Abstract
Neutrophils are immune cells that eliminate microbes using a powerful arsenal of reactive oxygen species, hypohalous acids, proteases, and antimicrobial peptides — yet how these cytotoxic effectors act in concert remains poorly understood. Here, we identify an unrecognized synergy between oxidative and non-oxidative neutrophil defenses. We show that myeloperoxidase-derived hypohalous acids selectively halogenate α-defensins (HNP1–3) at conserved tyrosine residues. This post-translational modification occurs in both human and rat neutrophils and is prominent in multiple diseases marked by neutrophil-rich inflammation. Halogenation increases HNP1 hydrophobicity without altering its structure, reprogramming these peptides into potent immunomodulatory mediators. Using single-cell transcriptomics and an in vivo peritonitis model, we demonstrate that halogenated HNP1s amplify immune signaling. Our analysis of the human haloproteome reveals a novel oxidative mechanism by which hypohalous acids rewire immune protein functions, uncovering an unexpected layer of cooperation between neutrophil effector systems. The discovery of halogenated HNP1s represents, to our knowledge, the first characterization of protein halogenation in human immunity.
Dataset DOI: https://doi.org/10.5061/dryad.02v6wwqkv
Description of the data and file structure
This dataset contains the underlying numerical values for every quantitative panel of the associated article. The measurements come from primary human neutrophils, monocytes and monocyte-derived macrophages isolated from the peripheral blood of healthy donors; from neutrophils and clinical specimens (serum, plasma, bronchoalveolar lavage, sputum, synovial fluid, abscess material) of patients; from rat and mouse neutrophils; and from chemically synthesised unmodified and halogenated α-defensin peptides.
The measurement types represented are: liquid-chromatography tandem mass spectrometry (LC-MS/MS) of halogenated peptides and proteins, electrospray-ionisation mass spectrometry (ESI-MS) of hydrolysed amino acids, plate-reader assays (fluorescence, absorbance, luminescence, LDH release, ELISA), thermal-shift and chromatographic peptide characterisation, NMR-derived chemical shifts, transwell chemotaxis counts, colony-forming-unit and microbial growth assays, flow cytometry counts, bulk RNA-sequencing and single-cell RNA-sequencing, and the functional-enrichment tables derived from those sequencing data.
All data are contained in a single Excel workbook, Data_S1.xlsx. Each worksheet holds the values for one panel of one figure and is named after that panel. Worksheets are described individually below. Panels of the article that consist only of images, structural models, gels, autoradiographs or spectra have no corresponding worksheet.
For full experimental procedures, instrument settings, peptide synthesis, and analysis pipelines, see the Materials and Methods and the Supplementary Information of the associated article.
Conventions used throughout the workbook
These apply to many worksheets and are not repeated in every entry below.
- Repeated column headers. Where the same header appears in two or more consecutive columns, those columns are replicates of that condition. Unless an entry states otherwise, they are independent biological replicates (separate donors or separate animals); within a single representative experiment they are technical replicates.
- Peptide nomenclature.
HNP1is unmodified human neutrophil peptide 1.ClY21-HNP1carries 3-chlorotyrosine at Tyr21,ClY16-HNP1at Tyr16,diClY21-HNP1is the dichlorinated Tyr21 form,IY16-HNP1andIY21-HNP1carry 3-iodotyrosine at Tyr16 and Tyr21.redprefixing a peptide name means the peptide was reduced with DTT and is used as a negative control.RNP1,ClY22-RNP1andIY22-RNP1are the corresponding rat neutrophil peptide 1 species. - Mass-spectrometry columns.
PEPis the posterior error probability of the peptide-spectrum match;Scoreis the Andromeda search-engine score;IntensityandLFQ intensityare MaxQuant raw and label-free-quantification peptide intensities in arbitrary units;genesholds the gene name(s) of the parent protein, with multiple names separated by semicolons where the peptide is shared between proteins. - Differential-expression columns. In all RNA-seq worksheets,
baseMeanis the mean normalised count across samples,log2FoldChangethe log2 fold change,lfcSEits standard error,statthe Wald statistic,pvaluethe raw andpadjthe Benjamini–Hochberg-adjusted p-value. - Empty cells mark replicates that were not performed or values below the detection limit, as stated per worksheet.
Worksheets and variables
Sheet: Fig. 1C
Description: ESI-MS quantification of 3-chlorotyrosine and total tyrosine in acid hydrolysates of human neutrophils left unstimulated or activated with PMA, S. aureus or zymosan. The four conditions repeat down the sheet, one block per independent donor.
Variables
- (first, unlabelled column) – stimulation condition: Unstim., PMA, S. aureus, Zymosan beads
- m/z 286 3-Cl-Tyr – integrated peak area of the 3-chlorotyrosine transition (arbitrary units)
- pmol 3-Cl-Tyr – 3-chlorotyrosine recovered, converted from peak area against a standard curve (pmol)
- m/z 352 Tyr – integrated peak area of the tyrosine transition (arbitrary units)
- pmol Tyr – tyrosine recovered (pmol)
- % Cl-Tyr/Tyr – chlorotyrosine as a percentage of total tyrosine
Sheet: Fig. 1E
Description: Relative abundance of the proteins recovered from the ~10 kDa radiolabelled gel band, measured by LC-MS proteomics. Ten proteins, three replicate columns each.
Variables
- HNP1-3, S100A9, HISTH3A, HIST1H4A, CSTA, S100A8, UBA52, HIST1H2BL, LOR, KPRP – relative protein abundance (% of total band intensity), three replicate columns per protein
Sheet: Fig. 1F
Description: Chlorinated peptides identified by LC-MS/MS in human neutrophils stimulated with PMA or S. aureus. One row per identified peptide.
Variables
- PEP – posterior error probability of the identification
- Score – Andromeda score
- Intensity – peptide intensity (arbitrary units)
- (fourth, unlabelled column) – gene name(s) of the parent protein
- (fifth, unlabelled column) – stimulus for the block of rows that follows (PMA or S. aureus)
Sheet: Fig. 1G
Description: Iodinated peptides identified by LC-MS/MS in human neutrophils stimulated with PMA or S. aureus. Layout as in Fig. 1F.
Variables
- score – Andromeda score
- PEP – posterior error probability
- intensity – peptide intensity (arbitrary units)
- genes – gene name(s) of the parent protein
- (fifth, unlabelled column) – stimulus for the block of rows that follows
Sheet: Fig. 1H
Description: Brominated peptides identified by LC-MS/MS in human neutrophils stimulated with PMA or S. aureus. Layout as in Fig. 1G.
Variables
- score – Andromeda score
- PEP – posterior error probability
- intensity – peptide intensity (arbitrary units); zero where the peptide was identified but not quantified
- genes – gene name(s) of the parent protein
- (fifth, unlabelled column) – stimulus for the block of rows that follows
Sheet: Fig. 1I
Description: Hypohalous-acid production measured with the APF fluorescent probe in human neutrophils, human monocytes, mouse neutrophils and rat neutrophils. The four cell types are repeated in three successive blocks of columns, one block per treatment (naive, H₂O₂, S. aureus). One row per independent experiment.
Variables
- experiment – anonymised experiment identifier (exp.1, exp.2, …), one per independent experiment
- human PMN, human MO, mouse PMN, rat PMN – APF fluorescence (arbitrary units), one column per cell type within each of the three treatment blocks
Sheet: Fig. 1J
Description: Chlorotyrosine measured by ESI-MS in the same four cell types, expressed as fold change over the matched naive control. Two blocks of columns, one per stimulus (H₂O₂, S. aureus).
Variables
- experiment – anonymised experiment identifier (exp.1, exp.2, …), one per independent experiment
- human PMN, human MO, mouse PMN, rat PMN – ClY fold change over naive (dimensionless), one column per cell type within each stimulus block
Sheet: Fig. 1M
Description: Chlorinated peptides identified by LC-MS/MS in rat neutrophils stimulated with S. aureus.
Variables
- PEP – posterior error probability
- Score – Andromeda score
- Intensity – peptide intensity (arbitrary units)
- genes – gene name(s) of the parent protein
Sheet: Fig. 1N
Description: Iodinated peptides identified by LC-MS/MS in rat neutrophils stimulated with S. aureus. Columns as in Fig. 1M.
Sheet: Fig. 1O
Description: Brominated peptides identified by LC-MS/MS in rat neutrophils stimulated with S. aureus. Columns as in Fig. 1M.
Sheet: Fig. 2A
Description: Luminol chemiluminescence time course reporting total ROS production in unstimulated and PMA-stimulated neutrophils from healthy donors, a myeloperoxidase-deficient patient and a chronic granulomatous disease patient. One row per successive read of the plate; three technical replicate columns per condition. Donor and patient assays were run in parallel on the same plate.
Variables
- HD unst, HD PMA, MPO patient PMA, MPO patient unst, CGD patient unst, CGD patient PMA – relative luminescence units, three replicate columns each
Sheet: Fig. 2B
Description: APF fluorescence time course reporting hypohalous-acid formation in the same donor and patient neutrophils, unstimulated or PMA-stimulated. Three technical replicate columns per condition.
Variables
- minute – time from stimulation (min)
- HD unst, HD PMA, CGD patient unst, CGD patient PMA, MPO patient unst, MPO patient PMA – APF fluorescence (arbitrary units), three replicate columns each
Sheet: Fig. 2C
Description: Chlorotyrosine-to-tyrosine ratio measured by acid hydrolysis and ESI-MS in neutrophils from healthy donors, a CGD patient and an MPO-deficient patient, with the patient cells additionally challenged with exogenous H₂O₂ or HOCl. One row per technical replicate.
Variables
- HD – ClY/Y ratio (%) in healthy-donor neutrophils
- CGD patient – ClY/Y ratio (%) in CGD neutrophils
- MPO patient – ClY/Y ratio (%) in MPO-deficient neutrophils
- CGD patient + exH2O2 – ClY/Y ratio (%) after exogenous hydrogen peroxide
- MPO patient + exHOCl – ClY/Y ratio (%) after exogenous hypochlorous acid
Sheet: Fig. 2D
Description: Prevalence of each halogenated HNP1 species across the clinical cohorts, as the percentage of patients in the cohort in whom that modification was detected. One row per cohort. Each modification occupies the first of three merged columns.
Variables
- (first, unlabelled column) – clinical cohort: neonatal sepsis, sepsis, pancreatitis, RA, cystic fibrosis, influenza, S. pneumoniae, L. pneumophila, S. aureus
- IY16, IY21, ClY21, ClY16, diClY21 – percentage of patients in that cohort positive for the modification (%)
Sheet: Fig. 2E
Description: Label-free quantification intensities of the three chlorinated HNP1 species, pooled across all patient samples in which each species was detected. One row per patient-sample observation; columns are not row-matched to one another.
Variables
- ClY21, diClY21, ClY16 – log₁₀ LFQ intensity of each chlorinated HNP1 species
Sheet: Fig. 2F
Description: LFQ intensities of the two iodinated HNP1 species, pooled across all patient samples in which each was detected. Layout as in Fig. 2E.
Variables
- IY16, IY21 – log₁₀ LFQ intensity of each iodinated HNP1 species
Sheet: Fig. 2G
Description: LFQ intensity of the doubly modified HNP1 peptide carrying IY16 and ClY21 on the same molecule, by cohort. One row per cohort; each value in the row is one patient.
Variables
- (first, unlabelled column) – clinical cohort
- IY16+ClY21 – log₁₀ LFQ intensity of the doubly modified peptide, one column per patient (trailing columns empty for smaller cohorts)
Sheet: Fig. 2H
Description: Per-patient LFQ intensities of IY16 and ClY21 on HNP1, by cohort. One row per cohort; eleven columns per modification, each holding one patient, with unused columns left empty.
Variables
- (first, unlabelled column) – clinical cohort: RA, sepsis, pancreatitis, S. aureus, influenza, neonatal sepsis, L. pneumophila
- IY16 – log₁₀ LFQ intensity of the Tyr16-iodinated peptide, one column per patient
- ClY21 – log₁₀ LFQ intensity of the Tyr21-chlorinated peptide, one column per patient
Sheet: Fig. 2I
Description: Chlorinated peptides identified by LC-MS/MS in sputum from cystic fibrosis patients.
Variables
- intesity (header as spelled in the file) – peptide intensity (arbitrary units)
- PEP – posterior error probability
- Score – Andromeda score
- genes – gene name(s) of the parent protein
Sheet: Fig. 2J
Description: Chlorinated peptides identified by LC-MS/MS in bronchoalveolar lavage from S. pneumoniae-infected patients.
Variables
- PEP – posterior error probability
- Score – Andromeda score
- intensity – peptide intensity (arbitrary units)
- (fourth, unlabelled column) – gene name(s) of the parent protein
Sheet: Fig. 2K
Description: Chlorinated peptides identified by LC-MS/MS in S. aureus skin abscess material.
Variables
- PEP – posterior error probability
- Score – Andromeda score
- Intensity – peptide intensity (arbitrary units)
- genes – gene name(s) of the parent protein
Sheet: Fig. 2L
Description: Iodinated peptides identified by LC-MS/MS in cystic fibrosis sputum.
Variables
- intesity (header as spelled in the file) – peptide intensity (arbitrary units)
- PEP – posterior error probability
- Score – Andromeda score
- genes – gene name(s) of the parent protein
Sheet: Fig. 2M
Description: Iodinated peptides identified by LC-MS/MS in S. pneumoniae bronchoalveolar lavage. Columns as in Fig. 2K.
Sheet: Fig. 2N
Description: Iodinated peptides identified by LC-MS/MS in S. aureus skin abscess material. Columns as in Fig. 2K.
Sheet: Fig. 3G
Description: Reverse-phase UPLC elution profiles of HNP1, ClY21-HNP1 and IY16-HNP1 on a C18 column. One row per acquisition point.
Variables
- control – retention time (min); the header is a legacy label, the values are the time axis shared by all three traces
- HNP1, ClY21-HNP1, IY16-HNP1 – absorbance (arbitrary units)
Sheet: Fig. 3H
Description: Nile red fluorescence of HNP1 and its halogenated forms, reporting exposed hydrophobic surface. One row per independent experiment.
Variables
- experiment – anonymised experiment identifier (exp.1, exp.2, …), one per independent experiment
- HNP1, ClY21-HNP1, IY21-HNP1 – Nile red fluorescence (arbitrary units)
Sheet: Fig. 3I
Description: Thermal-shift melting curves for the binding of anthrax Lethal Factor to HNP1 and its halogenated forms. One row per temperature step.
Variables
- Temperature [°C] – sample temperature
- HNP1, ClY21-HNP1, IY16-HNP1, LF – normalised fluorescence ratio (dimensionless); the trailing LF columns hold the replicate traces of the Lethal Factor control
Sheet: Fig. 3J
Description: Cleavage of a fluorogenic Lethal Factor substrate in the presence of HNP1 or its halogenated forms. One row per independent experiment.
Variables
- LF – substrate turnover with Lethal Factor alone (arbitrary fluorescence units)
- HNP1, ClY21-HNP1, IY16-HNP1 – substrate turnover in the presence of each peptide
Sheet: Fig. 3L
Description: Lethal Factor cytotoxicity on RAW 264.7 macrophages measured by LDH release, across a peptide concentration series. LL-37 is the negative control.
Variables
- concentr. HNP1 or LL-37 (uM) – peptide concentration (µM)
- HNP1, ClY21-HNP1, IY16-HNP1, LL-37 – LDH release (arbitrary units)
Sheet: Fig. 3M
Description: Listeriolysin O haemolysis of red blood cells across a peptide concentration series.
Variables
- concentr. HNP1 or LL-37 (uM) – peptide concentration (µM)
- HNP1, IY16-HNP1, ClY21-HNP1, LL-37 – haemolysis, measured as haemoglobin absorbance (arbitrary units)
Sheet: Fig. 3N
Description: Pneumolysin haemolysis of red blood cells across a peptide concentration series. Columns as in Fig. 3M.
Sheet: Fig. 3O
Description: Panton–Valentine leukocidin cytotoxicity on human neutrophils across a peptide concentration series. Columns as in Fig. 3M.
Sheet: Fig. 4 A-B
Description: Transwell chemotaxis counts. The sheet holds two stacked blocks: monocytes in the upper block and neutrophils in the lower block, each introduced by a label row and its own header row. One row per independent donor. CCL2 and fMLP are the positive controls for monocytes and neutrophils respectively; DTT-reduced peptides are negative controls.
Variables
- Untreated – migrated cells per field, vehicle control
- CCL2 (monocyte block) / fMLP (neutrophil block) – migrated cells, positive control
- HNP1, ClY21-HNP1, IY16-HNP1 – migrated cells per peptide
- red HNP1, red ClY21-HNP1, red IY16-HNP1 – migrated cells with DTT-reduced peptides
Sheet: Fig. 4D
Description: Full differential-expression table for HNP1-treated versus untreated human monocyte-derived macrophages (bulk RNA-seq). One row per gene, with normalised expression per replicate, group means, test statistics and gene annotation.
Variables
- gene_id – Ensembl gene identifier
- HNP1 – normalised expression in each of six HNP1-treated replicates
- untreated – normalised expression in each of six untreated replicates
- HNP1, untreated (single columns following the replicate blocks) – group mean normalised expression
- log2FoldChange, pvalue, padj – differential-expression statistics
- gene_name, gene_chr, gene_start, gene_end, gene_strand, gene_length, gene_biotype, gene_description, tf_family – gene annotation
- Sn_count – raw read count for sample n
- Sn_fpkm – FPKM-normalised expression for sample n
Sheet: Fig. 4E
Description: Differential-expression table for ClY21-HNP1 versus HNP1 in monocyte-derived macrophages. Column structure as in Fig. 4D, with the two replicate blocks labelled ClY21HNP1 and HNP1.
Sheet: Fig. 4F
Description: Differential-expression table for IY16-HNP1 versus untreated monocyte-derived macrophages. Column structure as in Fig. 4D, with the two replicate blocks labelled IY16-HNP1 and HNP1.
Sheet: Fig. 4G
Description: Top enriched GO biological-process terms for each pairwise comparison. The sheet holds three stacked blocks, each introduced by a label row naming the comparison (HNP1, ClY21-HNP1, IY16-HNP1), with the same nineteen terms in each block.
Variables
- GO term – name of the GO biological-process term
- Padj – adjusted enrichment p-value. Note that the exponents were lost when this column was exported: the digits match the corresponding GO tables in the Fig. S10 worksheets (for example 73.6956203306668 here against 7.36956203306668e-13 there), so read the values in those worksheets instead.
- Count – number of genes from the input set annotated to the term
Sheet: Fig. 5_A_umap_celltype
Description: Per-cell UMAP coordinates and reference-mapped cell-type labels for the single-cell RNA-seq of PBMCs treated with vehicle, HNP1, ClY21-HNP1 or IY16-HNP1. One row per cell.
Variables
- cell – unique cell barcode, prefixed with the sample identifier
- sample – sample identifier, encoding donor and condition
- treatment – experimental group: X (vehicle), UM (unmodified HNP1), Cl (ClY21-HNP1), I (IY16-HNP1)
- predicted.celltype.l1 – cell type transferred from the human PBMC reference atlas, level-1 granularity
- UMAP_1, UMAP_2 – UMAP embedding coordinates
Sheet: Fig. 5_B_deg_counts
Description: Number of significantly differentially expressed genes per cell type and per contrast in the single-cell data. One row per cell-type/contrast pair.
Variables
- cell_type – B, CD4 T, CD8 T, DC, Mono, NK, other T
- contrast – pairwise comparison, written as treatment_vs_treatment using the treatment codes above
- n_de_genes – count of genes with adjusted p-value < 0.05 (Wald test)
Sheet: Fig. 5_C_volcano_ClvsUM_Mono
Description: Pseudobulk differential-expression results in monocytes for ClY21-HNP1 versus unmodified HNP1. One row per gene.
Variables
- gene_name – HGNC gene symbol
- baseMean, log2FoldChange, lfcSE, stat, pvalue, padj – differential-expression statistics
- cell_type – cell type the test was run in (Mono)
- contrast – comparison tested (Cl_vs_UM)
Sheet: Fig. 5_D_volcano_IvsUM_Mono
Description: Pseudobulk differential expression in monocytes for IY16-HNP1 versus unmodified HNP1. Columns as in Fig. 5_C.
Sheet: Fig. 5_E_volcano_ClvsI_Mono
Description: Pseudobulk differential expression in monocytes for ClY21-HNP1 versus IY16-HNP1. Columns as in Fig. 5_C.
Sheet: Fig. 5_F_tmod_Mono
Description: Gene-set enrichment results in monocytes for the three peptide comparisons, from the tmod CERNO test. One row per enriched gene set.
Variables
- ID – gene-set identifier in the blood transcriptional module collection
- Title – gene-set name
- cerno – CERNO test statistic
- N1 – number of genes in the set present in the data
- AUC – area under the curve for the set
- cES – effect size
- P.Value, adj.P.Val – raw and adjusted enrichment p-values
- contrast – comparison tested
Sheet: Fig. 5_G_scatter_ClvsX_Mono
Description: Paired log2 fold changes in monocytes used to compare the ClY21-HNP1 and the unmodified-HNP1 responses, each against vehicle. One row per gene differentially expressed in at least one of the two comparisons.
Variables
- gene_name – HGNC gene symbol
- log2FC_x, padj_x – log2 fold change and adjusted p-value on the x-axis comparison
- log2FC_y, padj_y – log2 fold change and adjusted p-value on the y-axis comparison
- chemokine_cluster_ii – TRUE if the gene belongs to the chemokine cluster (II) gene set, FALSE otherwise
Sheet: Fig. 5_H_scatter_IvsX_Mono
Description: Paired log2 fold changes in monocytes for the IY16-HNP1 comparison. Columns as in Fig. 5_G.
Sheet: Fig. 5I
Description: Peritoneal monocyte infiltration in rats 24 h after intraperitoneal injection of each peptide, measured by flow cytometry. One row per animal.
Variables
- vehicle, RNP1, Cl-RNP1, I-RNP1, rCCL2 – CD11bhigh CD68+ cells as a percentage of total peritoneal cells (%)
Sheet: Fig. 5J
Description: Peritoneal granulocyte infiltration in the same animals, measured by flow cytometry. One row per animal.
Variables
- vehicle, RNP1, Cl-RNP1, I-RNP1, rCCL2 – RP1+ cells as a percentage of total peritoneal cells (%)
Sheet: Fig. S1A
Description: Hypohalous-acid production measured with APF in human neutrophils stimulated with PMA, S. aureus or zymosan, with or without the NADPH-oxidase inhibitor DPI or the MPO inhibitor 4-ABAH. One row per independent experiment.
Variables
- experiment – anonymised experiment identifier (exp.1, exp.2, …), one per independent experiment
- Untreated – APF fluorescence, unstimulated (arbitrary units)
- PMA, S. aureus, zymosan beads – APF fluorescence per stimulus
- PMA + DPI, S. aureus + DPI, zymosan beads + DPI – the same stimuli with DPI
- PMA + 4-ABAH, S. aureus + 4-ABAH, zymosan beads + 4-ABAH – the same stimuli with 4-ABAH
Sheet: Fig. S1B
Description: Chlorotyrosine-to-tyrosine ratio in neutrophils activated with physiological agonists, measured by mass spectrometry. One row per donor.
Variables
- naive – ClY/Tyr ratio (%) in unstimulated cells
- C5a, TNF + Heme, CXCL8 – ClY/Tyr ratio (%) after each agonist
Sheet: Fig. S1C
Description: Peak values from the ESI-MS elution profile of non-hydrolysed neutrophil lysates, with a chlorotyrosine standard for reference. A single row of values.
Variables
- PMA – signal from PMA-stimulated lysate (arbitrary units)
- unst. – signal from unstimulated lysate (arbitrary units)
- ClY standard – signal from the chlorotyrosine standard (arbitrary units)
Sheet: Fig. S1G
Description: UPLC quantification of chlorotyrosine and tyrosine in the ammonium-sulphate fractions of S. aureus-activated neutrophils, recorded as a laboratory worksheet rather than a tidy table. The upper rows document the sample-preparation steps and the calibration standards; the measured values follow below under their own header rows.
Variables
- (row labels in the first columns) – sample or standard identity, including the AQC blank and the calibration points
- Area /1000 – integrated UPLC peak area, divided by 1000, for 3-Cl-Tyr, Tyr and the Val internal standard
- pmol – amounts of 3-Cl-Tyr and Tyr derived from the peak areas (pmol)
Sheet: Fig. S1H
Description: The same UPLC quantification for PMA-activated neutrophils, laid out as in Fig. S1G, with one block per biological replicate.
Variables
- fraction – ammonium-sulphate fraction number (1–6)
- Area 3-Cl-Tyr, Area Tyr – integrated UPLC peak areas (arbitrary units)
- pmol 3-Cl-Tyr, pmol Tyr – amounts derived from the peak areas (pmol)
Sheet: Fig. S1I
Description: LC-MS quantification of the most abundant proteins in ammonium-sulphate fraction 1. One row per replicate.
Variables
- HNP1-3, S100A9, S100A8, ACTB, HBB, UNC79, DSC1, B0YJC4, INP4B, LYZ – protein intensity (arbitrary units); B0YJC4 is a UniProt accession for which no gene symbol was assigned
Sheet: Fig. S1J
Description: Chlorotyrosine content of each ammonium-sulphate fraction after SDS-PAGE separation and LC-MS/MS. A single row of values.
Variables
- AS fraction 1 … AS fraction 6 – chlorotyrosine signal per fraction (arbitrary units); zero indicates below detection
Sheet: Fig. S1L
Description: LC-MS intensities of chlorinated HNP1-derived peptides extracted from each band of a tricine gel of PMA-stimulated neutrophil lysate. One row per gel band; three technical replicate columns per peptide.
Variables
- band – gel band number (1–6)
- ClY-GTCIYQGR – intensity of the peptide chlorinated at Tyr16 (arbitrary units), three replicate columns
- YGTCI-ClY-QGR – intensity of the peptide chlorinated at Tyr21 (arbitrary units), three replicate columns
Sheet: Fig. S1M
Description: The same measurement for iodinated HNP1-derived peptides. Layout as in Fig. S1L.
Variables
- band – gel band number (1–6)
- IY-GTCIYQGR – intensity of the peptide iodinated at Tyr16, three replicate columns
- YGTCI-IY-QGR – intensity of the peptide iodinated at Tyr21, three replicate columns
Sheet: Fig. S2A
Description: Mass-spectrometry quantification of chlorinated HNP1-3 peptides from human neutrophils stimulated with PMA, with or without the MPO inhibitor 4-ABAH. One row per technical replicate.
Variables
- experiment – anonymised experiment identifier (exp.1, exp.2, …), one per independent experiment
- unst – peptide intensity in unstimulated cells (arbitrary units)
- PMA – peptide intensity after PMA
- PMA + 4-ABAH – peptide intensity after PMA in the presence of 4-ABAH
Sheet: Fig. S2B
Description: The same measurement for iodinated HNP1-3 peptides. Columns as in Fig. S2A.
Sheet: Fig. S2C
Description: LFQ intensities of the eighteen most abundant azurophilic-granule proteins, ranked by decreasing intensity. Three replicate columns per protein; values are entered in the first column of each triplet.
Variables
- Hours – incubation time label for the row
- HNP1-3, ELANE, MPO, CTSG, AZU1, PRTN3, RNASE3, EPX, CD63, BPI, LAMP2, LYZ, GRN, LTF, NPC2, RETN, LCN2, LAMP1, PSAP – LFQ intensity (arbitrary units), three columns per protein
Sheet: Fig. S2D
Description: Chlorinated peptides detected by LC-MS/MS in isolated azurophilic granules after a cell-free oxidative challenge with H₂O₂. A single row of values.
Variables
- HNP1, LYZ, MPO, ALB – intensity of the chlorinated peptide from each protein (arbitrary units); zero indicates not detected. The HNP1 cell holds the text string "10e+1543879" rather than a number; treat it as the highest value in the row and refer to the source data for the exact figure.
Sheet: Fig. S2E
Description: Total HNP1-3 measured by LC-MS/MS in the supernatant of naive and degranulating neutrophils. One row per replicate.
Variables
- naive PMN SN – HNP1-3 intensity in naive supernatant (arbitrary units)
- degranulating PMN SN – HNP1-3 intensity in degranulation supernatant (arbitrary units)
Sheet: Fig. S2F
Description: Chlorinated HNP1 (ClY21-HNP1) in NET fractions and in the matched supernatants of PMA- and nigericin-stimulated neutrophils. One row per donor.
Variables
- NETs PMA – ClY21-HNP1 intensity in the PMA NET fraction (arbitrary units)
- NETs Nigericin – ClY21-HNP1 intensity in the nigericin NET fraction; one donor was below the detection limit
- naive PMN supernatant, degranulating PMN supernatant – ClY21-HNP1 intensity in the supernatants; zero indicates not detectable
Sheet: Fig. S2G
Description: Chlorinated HNP1-3 as a percentage of total HNP1-3 in NETs. One row per donor.
Variables
- PMA, Nigericin – halogenated HNP1-3 as a percentage of total HNP1-3 (%)
Sheet: Fig. S2I
Description: Chlorinated peptides detected by LC-MS/MS in S. aureus-stimulated rat neutrophils, with RNP1 the most intense. A single row of values.
Variables
- RNP1, TTC21a, ZFP717, PCF11, ABCC2 – intensity of the chlorinated peptide from each protein (arbitrary units)
Sheet: Fig. S3E
Description: Relative abundance of the unmodified HNP1 tryptic peptide and its two halogenated forms in stimulated human neutrophils. A single row of values summing to 1.
Variables
- YGTCIYchQGR – relative abundance of the Tyr21-chlorinated peptide (fraction of the total peptide pool)
- YioGTCIYQGR – relative abundance of the Tyr16-iodinated peptide
- YGTCIYQGR – relative abundance of the unmodified peptide
Sheet: Fig. S3F
Description: Relative proportions of the two halogenated species within the modified fraction alone. A single row of values.
Variables
- YGTCIYchQGR – relative abundance of the Tyr21-chlorinated peptide within the modified fraction
- YioGTCIYQGR – relative abundance of the Tyr16-iodinated peptide within the modified fraction
Sheet: Fig. S3G
Description: Halogenated HNP1-3 as a percentage of total HNP1-3, per patient, in each clinical cohort. One row per cohort; eleven columns per modification, each holding one patient.
Variables
- (first, unlabelled column) – clinical cohort
- IY16 – log₁₀ intensity of the Tyr16-iodinated species, one column per patient
- ClY21 – log₁₀ intensity of the Tyr21-chlorinated species, one column per patient
Sheet: Fig. S3H
Description: Chlorinated HNP1 measured by LC-MS/MS in neutrophils isolated from sepsis patients and from healthy donors, naive or PMA-stimulated. A single row of values.
Variables
- HD PMN naive, HD PMN PMA – chlorinated-HNP1 intensity in healthy-donor neutrophils (arbitrary units)
- Sepsis PMN naive, Sepsis PMN PMA – chlorinated-HNP1 intensity in sepsis-patient neutrophils (arbitrary units)
Sheet: Fig. S4A
Description: Halogenated peptides identified by LC-MS/MS in cystic fibrosis sputum, filtered to an Andromeda score above 50. One row per modified site.
Variables
- Gene names – gene name(s) of the parent protein
- Positions within proteins – modified residue and its position in the protein sequence
- PEP – posterior error probability
- Score – Andromeda score
- modification – type of halogenation (chlorination, iodination, bromination)
Sheet: Fig. S4B
Description: Halogenated peptides identified in S. pneumoniae bronchoalveolar lavage. Columns as in Fig. S4A.
Sheet: Fig. S4C
Description: Halogenated peptides identified in S. aureus skin abscess material. Columns as in Fig. S4A.
Sheet: Fig. S4D
Description: Pathway-enrichment results for the halogenated proteins found in cystic fibrosis sputum. One row per enriched term; summary rows and member rows are distinguished by the GroupID column.
Variables
- GroupID – cluster identifier with a suffix marking the row as the cluster summary or a member term
- Category – source database: GO Biological Processes, Reactome, WikiPathways, and others
- Term – database identifier for the term
- Description – term name
- LogP – log₁₀ enrichment p-value
- Log(q-value) – log₁₀ multiple-testing-corrected q-value
- Genes – Entrez gene identifiers contributing to the term, comma-separated
- Symbols – corresponding gene symbols, comma-separated
- InTerm_InList – genes in the term and in the input list, as a ratio
Sheet: Fig. S4E
Description: Pathway-enrichment results for the halogenated proteins in S. pneumoniae lavage. Columns as in Fig. S4D.
Sheet: Fig. S4F
Description: Pathway-enrichment results for the halogenated proteins in S. aureus abscess material. Columns as in Fig. S4D.
Sheet: Fig. S5A
Description: Intensity of the chlorotyrosine-containing HNP1 peptide (ClY21) in neutrophils pre-incubated with NaBr or NaI and then stimulated with PMA. One row per replicate.
Variables
- unst, PMA – peptide intensity without added halide (arbitrary units)
- unst NaBr, PMA NaBr – peptide intensity with 1 mM NaBr
- unst NaI, PMA NaI – peptide intensity with 1 mM NaI
Sheet: Fig. S5B
Description: Intensity of the iodotyrosine-containing HNP1 peptide (IY16) under the same conditions. Columns as in Fig. S5A.
Sheet: Fig. S5F
Description: In vitro chlorination of wild-type HNP1 and the R24A mutant by NaOCl, measured as the intensity of the Tyr21-chlorinated peptide. A single row of values.
Variables
- HNP1 – peptide intensity, wild-type peptide without NaOCl (arbitrary units)
- HNP1 NaOCl – peptide intensity, wild-type peptide with NaOCl
- HNP1 R24A – peptide intensity, R24A mutant without NaOCl
- HNP1 R24A NaOCl – peptide intensity, R24A mutant with NaOCl
Sheet: Fig. S5G
Description: In vitro iodination of wild-type HNP1 and the R14A mutant by MPO with NaI, measured as the intensity of the Tyr16-iodinated peptide. A single row of values.
Variables
- HNP1 – peptide intensity, wild-type peptide untreated (arbitrary units)
- HNP1 MPO NaI – peptide intensity, wild-type peptide with MPO and NaI
- HNP1 R14A – peptide intensity, R14A mutant untreated
- HNP1 R14A MPO NaI – peptide intensity, R14A mutant with MPO and NaI
Sheet: Fig. S8A
Description: Thermal-shift melting curves for the binding of Panton–Valentine leukocidin to HNP1 and its halogenated forms. One row per temperature step; three replicate columns per peptide, with the measured trace in the first column of each triplet.
Variables
- Temperature [°C] – sample temperature
- HNP1, ClY21-HNP1, IY16-HNP1, PVL – normalised fluorescence ratio (dimensionless), three columns each
Sheet: Fig. S8B
Description: Inhibition of S. aureus HlgAB-induced neutrophil death by HNP1 and its halogenated forms, measured by LDH release at three peptide concentrations. One row per peptide; three replicate columns per concentration.
Variables
- (first, unlabelled column) – peptide tested: HNP1, ClY21-HNP1, IY16-HNP1
- 0.1 µM, 1 µM, 10 µM – cell viability as a percentage of the untreated control (%), three replicate columns per concentration
Sheet: Fig. S8C
Description: The same assay with the HlgCB toxin. Columns as in Fig. S8B.
Sheet: Fig. S8D
Description: The same assay with the LukAB CC45 toxin. Columns as in Fig. S8B.
Sheet: Fig. S8E
Description: Metabolic activity of human monocytes exposed to Clostridioides difficile toxin TcdB in the presence of each peptide. One row per replicate.
Variables
- Untreated – metabolic activity of unexposed cells (absorbance, arbitrary units)
- TcdB – metabolic activity with toxin alone
-
* HNP1, + ClY21-HNP1, + IY16-HNP1 – metabolic activity with toxin plus each peptide
Sheet: Fig. S8F
Description: Direct cytotoxicity of each peptide on healthy-donor PBMCs, measured by LDH release across a concentration series. One row per donor. Triton X-100 gives maximum release.
Variables
- naive – LDH release from untreated cells (arbitrary units)
- triton 0.1% – LDH release after full lysis
- HNP1 1 µg/ml, HNP1 10 µg/ml, HNP1 100 µg/ml – LDH release per HNP1 concentration
- ClY21-HNP1 1 / 10 / 100 µg/ml – LDH release per ClY21-HNP1 concentration
- IY16-HNP1 1 / 10 / 100 µg/ml – LDH release per IY16-HNP1 concentration
Sheet: Fig. S8G
Description: Protease activity of neutrophil elastase, cathepsin G, MMP9 and proteinase K measured against fluorogenic substrates after pre-incubation with each peptide. One row per replicate. Each protease has its own specific inhibitor as a control.
Variables
- NE, NE + HNP1, NE + ClY21-HNP1, NE + IY16-HNP1, NE+NEi – elastase activity (% of uninhibited control)
- CG, CG + HNP1, CG + ClY21-HNP1, CG+ IY16-HNP1, CG + CGi – cathepsin G activity (%)
- MMP9, MMP9 + HNP1, MMP9 + ClY21-HNP1, MMP9+ IY16-HNP1, MMP9 + MMP9i – MMP9 activity (%)
- Proteinase K and its four counterpart columns – proteinase K activity (%)
Sheet: Fig. S9A
Description: Colony-forming units of S. aureus after incubation with each peptide at 50 µg/ml. DTT-reduced peptides are negative controls. One row per independent experiment.
Variables
- experiment – anonymised experiment identifier (exp.1, exp.2, …), one per independent experiment
- Untreated – CFU recovered without peptide (×10³ CFU/ml)
- HNP1, ClY21-HNP1, IY16-HNP1 – CFU recovered per peptide
- red HNP1, red ClY21-HNP1, red IY16-HNP1 – CFU recovered with DTT-reduced peptides
Sheet: Fig. S9B
Description: The same assay with Escherichia coli.
Variables
- experiment – anonymised experiment identifier (exp.1, exp.2, …), one per independent experiment
- Untreated, HNP1, ClY21-HNP1, IY16-HNP1 – CFU recovered (×10³ CFU/ml)
Sheet: Fig. S9C
Description: The same assay with Candida albicans. Columns as in Fig. S9B.
Sheet: Fig. S9D
Description: Growth curves of S. aureus in RPMI in the presence of each peptide, read on a 96-well plate. One row per read.
Variables
- Time – elapsed time from the start of the run (hh:mm:ss)
- (remaining columns) – optical density per well, in plate-reader well order; see the plate layout in the Supplementary Information for the condition assigned to each well
Sheet: Fig. S9E
Description: Growth curves of S. aureus in TSB in the presence of each peptide. One row per read.
Variables
- Time – elapsed time from the start of the run (hh:mm:ss)
- A1 … H12 – optical density per well, labelled by plate position; see the plate layout in the Supplementary Information for the condition assigned to each well
Sheet: Fig. S9F
Description: XTT metabolic-activity assay of C. albicans grown in the presence of each peptide. One row per time point; three replicate columns per condition.
Variables
- Time (hr) – elapsed time (h)
- blank – medium-only control (absorbance)
- yeast alone + vehicle – untreated yeast
- defensin alone – peptide without yeast
- wt defensin – unmodified HNP1
- Y16 Cl defensin – HNP1 chlorinated at Tyr16
- Y16, Y21 Cl defensin – doubly chlorinated HNP1
- wt defensin DTT, Y16 Cl defensin DTT, Y16, Y21 Cl defensin DTT – the same peptides reduced with DTT
Sheet: Fig. S9G
Description: Survival of S. aureus after incubation with neutrophils primed with each peptide. One row per healthy donor.
Variables
- Mock – bacterial survival with unprimed neutrophils (% of inoculum)
- GM-CSF – survival with GM-CSF-primed neutrophils
- UM – survival with neutrophils primed with unmodified HNP1
- ClY21, IY16 – survival with neutrophils primed with each halogenated peptide
Sheet: Fig. S9H
Description: Phagocytosis of C. albicans by monocyte-derived macrophages pre-treated with each peptide, 4 h after infection. One row per replicate.
Variables
- Untreated – phagocytic index without peptide
- HNP1, Cl-HNP1, I-HNP1 – phagocytic index per peptide
- red HNP1, red Cl-HNP1, red I-HNP1 – phagocytic index with DTT-reduced peptides
Sheet: Fig. S9J
Description: CCL2 released into the medium by monocyte-derived macrophages after 4 h of stimulation, measured by ELISA. One row per donor.
Variables
- naive – CCL2 concentration without stimulus (pg/ml)
- HNP1, ClY21-HNP1, IY16-HNP1 – CCL2 concentration per peptide
- red HNP1, red ClY21-HNP1, red IY16-HNP1 – CCL2 concentration with DTT-reduced peptides
Sheet: Fig. S9K
Description: CCL3 released under the same conditions, measured by ELISA. Columns as in Fig. S9J.
Sheet: Fig. S10A
Description: GO enrichment results for the genes upregulated by HNP1 relative to untreated macrophages. The comparison is named in the first row; the column headers are in the second row, one row per enriched term below.
Variables
- Category – ontology: BP (biological process), MF (molecular function) or CC (cellular component)
- GOID – GO term identifier
- Description – GO term name
- GeneRatio – genes from the input set in the term, over the input set size
- BgRatio – genes in the term, over the background set size
- pvalue, padj – raw and Benjamini–Hochberg-adjusted enrichment p-values
- geneID – Ensembl identifiers of the contributing genes, slash-separated
- geneName – corresponding gene symbols, slash-separated
- Count – number of contributing genes
- Up, Up_Gene_names – number and Ensembl identifiers of the upregulated contributing genes
- Down, Down_Gene_names – number and Ensembl identifiers of the downregulated contributing genes
Sheet: Fig. S10B
Description: GO enrichment results for ClY21-HNP1 versus HNP1. Columns as in Fig. S10A.
Sheet: Fig. S10C
Description: GO enrichment results for IY16-HNP1 versus untreated. Columns as in Fig. S10A.
Sheet: Fig. S15A(I)
Description: Differential-expression table for HNP1 versus untreated, in the replicate-labelled format used for the supplementary bulk RNA-seq analyses. The comparison is named in the first row; column headers are in the second row, one row per gene below.
Variables
- gene_id – Ensembl gene identifier
- AII, BII, CII – normalised expression in the three HNP1-treated replicates
- AI, BI, CI – normalised expression in the three untreated replicates
- HNP1, untreated – group mean normalised expression
- log2FoldChange, pvalue, padj – differential-expression statistics
- gene_name, gene_chr, gene_start, gene_end, gene_strand, gene_length, gene_biotype, gene_description, tf_family – gene annotation
- AII_count … CI_count – raw read counts per replicate
- AII_fpkm … CI_fpkm – FPKM-normalised expression per replicate
Sheet: Fig. S15A(II)
Description: Differential-expression table for ClY21-HNP1 versus HNP1, with replicate columns AIII, BIII, CIII (ClY21-HNP1) against AII, BII, CII (HNP1). Column structure as in Fig. S15A(I).
Sheet: Fig. S15A(III)
Description: Differential-expression table for IY16-HNP1 versus HNP1, with replicate columns AIV, BIV, CIV (IY16-HNP1) against AII, BII, CII (HNP1). Column structure as in Fig. S15A(I).
Sheet: Fig. S15B
Description: GO enrichment results for HNP1 versus untreated in this supplementary analysis. Columns as in Fig. S10A.
Sheet: Fig. S15C
Description: GO enrichment results for ClY16-HNP1 versus HNP1. Columns as in Fig. S10A.
Sheet: Fig. S15D
Description: GO enrichment results for IY16-HNP1 versus HNP1. Columns as in Fig. S10A.
Sheet: Fig.S11_A_pca_donor3_included
Description: Principal-component coordinates from the pseudobulk quality-control analysis of the single-cell data, with all three donors included. One row per sample.
Variables
- PC1, PC2 – principal-component coordinates
- group – condition and donor, written as condition:donor
- treatment – experimental group: X (vehicle), UM, Cl, I
- batch – donor number
- name – sample label, combining cell type and sample identifier
Sheet: Fig.S11_B_qc_metrics
Description: Per-cell quality-control metrics for the single-cell dataset. One row per cell.
Variables
- sample – sample identifier, encoding donor and condition
- nCount_RNA – total transcripts detected in the cell
- nFeature_RNA – genes detected in the cell
- predicted.celltype.l1.score, predicted.celltype.l2.score – reference-mapping label-transfer confidence at each level of granularity (0–1)
- sample_n – sample identifier with its total cell count appended
Sheet: Fig.S11_C_celltype_pct
Description: Cell-type composition of each sample. One row per sample and cell type.
Variables
- sample – sample identifier
- group – experimental group
- predicted.celltype.l1 – reference-mapped cell type
- n – number of cells of that type in the sample
- pct.cells – that type as a percentage of the sample (%)
Sheet: Fig.S11_D_marker_dotplot
Description: Scaled average expression of selected marker genes across the annotated cell types. One row per gene and cell type.
Variables
- avg.exp – mean normalised expression of the gene in that cell type
- pct.exp – percentage of cells of that type in which the gene was detected (%)
- features.plot – gene symbol
- id – cell type
- avg.exp.scaled – mean expression, z-scored across cell types
Sheet: Fig.S11_E_pca_final
Description: Principal-component coordinates from the pseudobulk analysis after the outlying donor was excluded. Columns as in Fig.S11_A.
Sheet: Fig.S12_A_tmod_UMvsX
Description: Gene-set enrichment results across all cell types for unmodified HNP1 versus vehicle. One row per enriched gene set.
Variables
- ID – gene-set identifier
- Title – gene-set name
- cerno – CERNO test statistic
- N1 – genes in the set present in the data
- AUC – area under the curve
- cES – effect size
- P.Value, adj.P.Val – raw and adjusted enrichment p-values
- cell_type – cell type in which the set was tested
Sheet: Fig.S12_B_tmod_ClvsX
Description: Gene-set enrichment for ClY21-HNP1 versus vehicle. Columns as in Fig.S12_A.
Sheet: Fig.S12_C_tmod_IvsX
Description: Gene-set enrichment for IY16-HNP1 versus vehicle. Columns as in Fig.S12_A.
Sheet: Fig.S13_A_upset_Mono
Description: Sizes of the intersections between the differentially expressed gene sets of the different contrasts, as plotted in the upset diagram. One row per intersection.
Variables
- combination – the contrast or combination of contrasts the intersection represents
- n – number of genes in that intersection
Sheet: Fig.S13B_volcano_vsvehicle_Mono
Description: Pseudobulk differential-expression results in monocytes for all three peptides against vehicle, stacked in one table. One row per gene and contrast.
Variables
- gene_name – HGNC gene symbol
- baseMean, log2FoldChange, lfcSE, stat, pvalue, padj – differential-expression statistics
- cell_type – cell type tested (Mono)
- contrast – comparison tested: UM_vs_X, Cl_vs_X or I_vs_X
Sheet: Fig.S14_A_scatter_ClvsUM_CD4T
Description: Paired log2 fold changes in CD4 T cells for the ClY21-HNP1 and unmodified-HNP1 responses, each against vehicle. One row per gene differentially expressed in at least one of the two comparisons.
Variables
- gene_name – HGNC gene symbol
- log2FC_x, padj_x – log2 fold change and adjusted p-value on the x-axis comparison
- log2FC_y, padj_y – log2 fold change and adjusted p-value on the y-axis comparison
Sheet: Fig.S14_B_scatter_IvsUM_CD4T
Description: The same paired comparison for IY16-HNP1 against unmodified HNP1. Columns as in Fig.S14_A.
Sheet: Fig.S14_C_scatter_ClvsI_CD4T
Description: The same paired comparison for ClY21-HNP1 against IY16-HNP1. Columns as in Fig.S14_A.
Code/software
All data are provided in a single Excel workbook (.xlsx), readable with Microsoft Excel 2007 or later, LibreOffice Calc, or any library that reads the Office Open XML format (for example openpyxl for Python or readxl for R). No custom code is required to open the files.
The derived tables in this workbook were produced with [MaxQuant vX.X] for the mass-spectrometry searches and label-free quantification, [DESeq2 vX.X] for differential expression, [clusterProfiler vX.X] and [tmod vX.X] for functional enrichment, and [Seurat vX.X] for the single-cell processing and reference mapping. [State here whether analysis scripts are deposited elsewhere, with the repository and accession if so.]
Human subjects data
Human samples were collected according to the Declaration of Helsinki, with study participants providing written informed consent. CGD and MPO patient samples were collected with approval from the ethics committee Charité-Universitätsmedizin Berlin. The patients with X-linked chronic granulomatous disease (CGD) harbor mutations in the NADPH subunit genes (CYBB c.742dupA, CYBB c.868C > T)46, 47. The ∆MPO donor bears a homozygous splice mutation (c.2031--‐ 2A >C/c. 20312 A>C, nomenclature according to(49), which generates null alleles that result in an immature protein(50). Patients from the sepsis and ARDS Registry (SPARE-14, Hannover Medical School) were treated in the intensive care unit and intubated for acute respiratory failure. Informed consent for biomaterial sampling was given by the patient or a legal representative before intubation (#8146_BO_K_2018). BAL was performed preferably in the middle lobe or lingual or radiologically affected area. BAL samples were centrifuged at 500 × g for 10min at 4°C, and the supernatant (BAL fluid) was stored at -80°C until use. Cystic Fibrosis patients were treated at the Charité-Universitätsmedizin Berlin and gave informed consent for the approved studies: BBL01a0041; RBB07a0039; RBB07a0373; RBB07a0371; RBB07a0414; RBB07a0469S.
Sputum samples were collected after spontaneous expectoration or induced with inhaled hypertonic saline (sodium chloride 6%). Samples were cooled and processed within 24 hours. Pus samples from S. aureus-infected patients were obtained with the approval and guidelines of the Charité-Universitätsmedizin Berlin ethics committee (EA2/003/019). A 4-5 mm punch biopsy was taken from the abscess under local anesthesia.
Informed consent was obtained from all subjects. Sepsis fresh samples were obtained from participants enrolled at Charité-Universitätsmedizin Berlin and approved by the Ethics Committee of Charité-Universitätsmedizin Berlin (EA4/255/23). The serum and plasma sepsis samples were obtained from CAPNEZ, and the study was conducted in accordance with the Declaration of Helsinki, as well as guidelines of Good Clinical Practice. It was approved by the institutional ethics board of the Hannover Medical School, Germany (Ethics approval No. 301-2008). BAL samples from patients with pneumonia, pancreatitis, and Legionella infection were obtained from the SPARE-14 registry at Hannover Medical School. Protocols and usage of human neonatal sepsis serum samples were approved by the Institutional Review Boards of the Hannover Medical School (no. 8014_BO_S_2018). These samples were obtained from five preterm infants (<33 weeks’ gestational age) diagnosed with late- onset neonatal sepsis (LON); two of them were blood culture-confirmed (Staphylococcus capitis, n = 1; Klebsiella aerogenes, n = 1). LONS diagnosis was based on ≥2 clinical signs of systemic inflammatory response plus ≥1 laboratory criterion. Clinical signs included temperature >38 °C or <36.5 °C, tachycardia >200 bpm, new or increased bradycardias or apneas, hemodynamic instability, hyperglycemia >10 mM, metabolic acidosis (base excess <–10 mEq/L), grayish skin color, and increased oxygen requirement. Laboratory signs included C-reactive protein >10 mg/L, IL-6 >150 ng/L, platelet count <100/nL, immature/total neutrophil ratio >0.2, granulocytopenia <1,000/µL, and white blood cell count <5/nL. Blood culture–confirmed sepsis was defined as clinical sepsis with bacterial growth in a blood culture obtained prior to antibiotic therapy. Protocols and usage of human neonatal sepsis biomaterial were approved by the Institutional Review Boards of the Hannover Medical School (no. 8014_BO_S_2018). Written informed consent was obtained from all participating individuals and the parents or legal representatives on behalf of the children enrolled in our study. Rheumatoid arthritis synovial fluid was obtained during routine clinical arthrocentesis under ethical approval from Charité-Universitätsmedizin Berlin (EA1/002/24).
All patients or legal representatives provided informed consent in accordance with the Declaration of Helsinki. Healthy donor samples (N = 5) served as controls for all comparisons. Halogenated peptides were enriched and quantified by high-resolution LC-MS/MS using a targeted acquisition method for chlorotyrosine (+33.96 Da) and iodotyrosine (+125.90 Da) modifications as described.
Written informed consent was obtained from all healthy donors and patients prior to sample collection, including consent for the deposition and secondary use of de-identified data.
De-identification. Donors and patients appear in this dataset only as sequential codes or as anonymous replicate columns. The workbook contains no names, initials, dates of birth, admission, sampling or experiment dates, hospital or laboratory record numbers, geographic information, or any other direct identifier. Patients are grouped only by clinical cohort (for example, "sepsis", "cystic fibrosis", "influenza"). Any key linking participant codes to identities is held exclusively by the recruiting institutions named above, under their respective approved protocols, and does not form part of this deposit.
Measures to reduce re-identification risk. All calendar dates originally used as experiment labels have been removed and replaced by sequential anonymous identifiers (exp.1, exp.2, …), or deleted where the label carried no information; no temporal sequence in the data depends on the true dates. No demographic variables (age, sex, ethnicity, place of treatment) are included, and no clinical metadata beyond the cohort label are reported. Cohort sizes are small for some diseases, so cohort membership is the only patient-level attribute present and is not combined with any other attribute that could narrow an individual down. The deposited worksheets contain experimental readouts only.