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Aliquots of each sample (containing 20 µg protein) were added to an equal volume of nitrogen-purged urea (8 M) in Tris buffer (100 mM, pH 8.5) and were reduced and alkylated by the addition of DTT (5 mM) with incubation for 30 min at 21 °C, followed by addition of iodoacetamide (10 mM) and a further 30 min incubation in the dark. Proteins were enzymatically digested using an SP3-based approach, as previously described (Hughes, C. S. et al., 2019, Nature protocols, 14(1), 68–85. DOI: 10.1038/s41596-018-0082-x). Samples (220 µL) were mixed with an equal volume of ethanol before addition of a solution of magnetic beads (1:55 ratio beads to sample; mix of 2 different Sera-Mag Magnetic Carboxylate Modified Particles, Lot: 44152105050250 and 24152105050250 in a 50:50 ratio; Cytiva) with incubation for 30 min in a thermomixer at 24 °C and 1000 rpm. The supernatant was removed after placing the samples on a magnet, followed by washing with 80 % v/v ethanol, with tubes placed on magnet between washes to enable removal of the supernatant. This step was repeated twice before removing the magnet and adding 100 μL trypsin digestion buffer (100 mM Tris buffer, pH 8) and 1 μL sequencing grade trypsin (0.1 μg/μL in 10 mM HCl; Promega), followed by overnight incubation at 21 °C. Trypsin-generated proteolytic peptides were isolated by a stage tipping approach based on Rappsilber et al. (Rappsilber, J. et al., 2007, Nature protocols, 2(8), 1896–1906. DOI: 10.1038/nprot.2007.261). Filter AttractSPETM Disks Bio - C18 (AFFINISEP) were activated with acetonitrile, then conditioned with 80 % v/v acetonitrile containing 0.1 % v/v trifluoroacetic acid (TFA) before equilibration with 0.1 % v/v TFA. Between these steps, the filter tips with tubes were centrifuged at 21 °C (1200 g for 1 min). The digested samples (20 µg peptides) were acidified with 10 % v/v TFA to reach a pH < 2 and loaded on the activated filter tips, with centrifugation (1200 g for 1 min). The filters with the peptides were washed twice with 0.1 % v/v TFA with centrifugation between washing steps (1200 g for 1 min). The peptides were eluted with 80 % v/v acetonitrile containing 0.1 % v/v formic acid into fresh tubes by centrifugation (1200 g for 1 min) before drying using a Speedvac (Refrigerated Condensation Trap RT490, Thermo Fisher). Prior to MS analysis, samples were reconstituted in 20 μL 0.1 % v/v formic acid to final concentration of 1 µg µL-1 digested protein. Elastase peptide samples were analyzed by LC-MS on an Impact II ESI-QTOF (Bruker Daltonics) mass spectrometer in the positive ion mode with an Apollo ion source connected on-line to a Dionex Ultimate 3000 chromatography system (Thermo Fisher) with a NanoLC pump. Analytes were separated using an 8 cm x 75 µm Aurora column (Ionoptics) at 40 °C with a flow rate of 400 nL/min and gradient elution (3-15, 15-44 and 44-99 % Solvent B, over 3, 17, 1 min, respectively using 0.1% formic acid (Solvent A) and 80% acetonitrile/0.1% formic acid (Solvent B). LC-MS/MS data were acquired using a data-dependent method with a MS scan followed by MS/MS scans with a cycle time of 1.5 sec.PrideThe MS data was searched against Chymotrypsin-like elastase family member 1 from pig reference UniProt proteome (P00772) without the signal sequence (1-16 amino acids) and the propeptide (17-26 amino acids), using FragPipe (version 22.0). The following parameters were used for the search: 2 missed tryptic cleavages; 30 ppm parent and fragment mass tolerance; 1% FDR, fixed modification: IAM alkylation of Cys (+57.02146 Da), variable modifications: oxidation (+15.9949 Da) and dioxidation (+31.9898 Da) of Met, Cys and Trp, trioxidation of Cys (+47.9847 Da) nitrile formation on Lys (-4.0313 Da), chlorination (+ 33.9610 Da) and dichlorination (+67.9221 Da) of Tyr and Trp, semialdehyde formation on Lys (-1.0316 Da), kynurenine formation of Trp (+3.9949 Da) and dihydroxy-Trp or N-formylkynurenine (NFK) formation on Trp (+31.9898 Da).ProteomicsClare Louise HawkinsPARTIALUniversity of Copenhagen, Faculty of Health and Medical Sciences, Department of Biomedical SciencesSus Scrofa Domesticus (domestic Pig)10.1016/J.FREERADBIOMED.2026.06.033helen@sund.ku.dkUniversity of Copenhagen, Faculty of Health and Medical Sciences, Department of Biomedical SciencesDenmarkfalseComparison of the reactivity of hypochlorous acid (HOCl) and hyposelenocyanous acid (HOSeCN) with elastase: role of amino acid modification and structural changes in loss of activity.Elastase is a serine protease that plays a key role in intracellular and extracellular neutrophil-driven immune responses. There is a synergistic relationship between elastase and myeloperoxidase (MPO), which can boost pathogen killing and limit collateral proteolytic damage to host tissue. However, both proteins are strongly implicated in disease pathology, leading to interest in the design of therapeutic strategies to modulate their activity in vivo, particularly in chronic inflammatory settings. In this study, we examine whether the alternative MPO substrate selenocyanate (SeCN-), can modulate the modification of elastase by hypochlorous acid (HOCl), by the favouring the formation of hyposelenocyanous acid (HOSeCN), as a potential therapeutic strategy. Exposure of elastase to HOCl results in significant loss of function, amino acid modification, fragmentation and aggregation. Trp and Tyr are readily modified, forming hydroxytryptophan derivatives, kynurenine and 3-chlorotyrosine, respectively. Loss of cystine, Met, His and Arg was also observed, together with the formation of N-chloramines, which did not appear to cause secondary oxidation. Inactivation of elastase was observed on exposure to HOSeCN, which correlated with strongly with unfolding. With HOSeCN, there was less extensive modification and loss of amino acid residues, with Trp and cystine residues shown to be the main targets. Fragmentation of elastase was observed under reducing conditions, shown by the presence of multiple low molecular mass bands, whereas a band at 50 kDa was seen in the absence of reducing agent, suggesting formation of a dimer. Supplementation of HOCl with sub-stoichiometric amounts of SeCN- prevented elastase inactivation but resulted in structural changes to elastase consistent with HOSeCN formation.2026-06-222026-03-11PXD075502NEWT:6945NEWT:3555NEWT:241368NEWT:2NEWT:157546NEWT:190802NEWT:35554NEWT:150475NEWT:9417NEWT:347515NEWT:1216979NEWT:307972NEWT:544496NEWT:5180NEWT:256737NEWT:115104NEWT:1081927NEWT:67825NEWT:13076NEWT:1249668NEWT:376741NEWT:317NEWT:1736309NEWT:7227NEWT:7469NEWT:885318NEWT:876138NEWT:4081NEWT:554NEWT:98334NEWT:237561NEWT:10036NEWT:7574NEWT:1351NEWT:7215NEWT:272563NEWT:507601NCBITaxon:79857NCBITaxon:6157NEWT:95648NEWT:746360NEWT:6239NEWT:1589NEWT:470150NEWT:135622NEWT:216257NEWT:6915NEWT:9986NEWT:101510NEWT:4054NEWT:3880NEWT:8782NEWT:1000589NEWT:1902NEWT:85962NEWT:160488NEWT:28104NEWT:317447NEWT:7955NCBITaxon:2NEWT:985076NEWT:7959NEWT:2261NEWT:4565NEWT:1264690NEWT:6192NEWT:28532NCBITaxon:38727NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:216595NEWT:243230NEWT:8355NEWT:931281NEWT:7029NEWT:1283300NEWT:334747NEWT:61235NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:3218NEWT:5759NEWT:1736231NEWT:436486NEWT:6287NEWT:2242NEWT:300641NEWT:4784NEWT:727NEWT:9796NEWT:725NEWT:360106NEWT:260707NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1836NEWT:1735272NEWT:83334NEWT:83332NEWT:29760NEWT:703612NEWT:260705NEWT:80863NEWT:44685NEWT:2697049NEWT:1148NEWT:11676NEWT:55571NEWT:100226NCBITaxon:6073NEWT:4530NEWT:4896NEWT:6279NEWT:7370NEWT:6282NEWT:1134506NEWT:575584NEWT:1773NEWT:38783NEWT:8727NEWT:1895NEWT:1182590NEWT:8726NEWT:10090NEWT:935293NEWT:749200NEWT:4120NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:990346NEWT:5334NEWT:145953NEWT:257309NEWT:230741NEWT:284812NCBITaxon:10359NCBITaxon:1313NEWT:43330NEWT:242619NEWT:44544NEWT:373995NEWT:5689NEWT:544404NEWT:3702NEWT:129249NEWT:8839NEWT:4232NEWT:990119NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:196627NEWT:408172NEWT:5691NEWT:408170NEWT:493760NEWT:260710NEWT:627025NEWT:400772NEWT:1097677NEWT:3708NEWT:106592NEWT:9913NEWT:1432138NEWT:10312NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:8030NEWT:29722NEWT:380394NEWT:1692259NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:135588NEWT:1843183NEWT:95486NEWT:58002NEWT:9103NEWT:4577NEWT:5664NEWT:2157NEWT:146479NEWT:10306NEWT:1911079NEWT:8022NEWT:145943NEWT:3635NEWT:5811NEWT:235443NEWT:1480154NEWT:1274414NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:884019NEWT:169963NEWT:36329NEWT:9606NEWT:367830NEWT:157295NEWT:410289NEWT:373153NEWT:915099NEWT:74940NEWT:1450511NEWT:470NEWT:84023NEWT:9838NCBITaxon:9615NEWT:1193501NEWT:3055NEWT:6326NEWT:6689NEWT:2762NEWT:5476NEWT:1174673NEWT:562NEWT:1274432NEWT:1274426NEWT:1423NEWT:4932NEWT:70448NEWT:9825NEWT:1274423NEWT:3603NEWT:698936NEWT:2759NEWT:3847NEWT:39946NEWT:9823NEWT:9940NEWT:573NEWT:9031NEWT:1274420NEWT:70910009-0008-2062-0115