{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T22:01:25Z","timestamp":1768341685930,"version":"3.49.0"},"reference-count":52,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,3,11]],"date-time":"2021-03-11T00:00:00Z","timestamp":1615420800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003130","name":"Fonds Wetenschappelijk Onderzoek","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004497","name":"Onderzoeksraad, KU Leuven","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004497","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010661","name":"Horizon 2020 Framework Programme","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100010661","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography\u2014tandem mass spectrometry (nano-LC-MS\/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and \u2013activating properties, occurs in different NH<jats:sub>2<\/jats:sub>-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.<\/jats:p>","DOI":"10.3389\/fimmu.2021.644725","type":"journal-article","created":{"date-parts":[[2021,3,11]],"date-time":"2021-03-11T06:04:56Z","timestamp":1615442696000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":12,"title":["From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8\/Interleukin-8"],"prefix":"10.3389","volume":"12","author":[{"given":"Mieke","family":"Metzemaekers","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sara","family":"Abouelasrar Salama","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer","family":"Vandooren","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anneleen","family":"Mortier","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rik","family":"Janssens","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sofie","family":"Vandendriessche","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eva","family":"Ganseman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Erik","family":"Martens","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mieke","family":"Gouwy","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Barbara","family":"Neerinckx","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick","family":"Verschueren","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lien","family":"De Somer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carine","family":"Wouters","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sofie","family":"Struyf","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ghislain","family":"Opdenakker","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jo","family":"Van Damme","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul","family":"Proost","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2021,3,11]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"182","DOI":"10.1016\/S0167-5699(97)01243-7","article-title":"The molecular basis of leukocytosis","volume":"19","author":"Opdenakker","year":"1998","journal-title":"Immunol Today."},{"key":"B2","doi-asserted-by":"publisher","first-page":"1223","DOI":"10.1152\/physrev.00012.2018","article-title":"The neutrophil's role during health and disease","volume":"99","author":"Liew","year":"2019","journal-title":"Physiol Rev."},{"key":"B3","doi-asserted-by":"publisher","first-page":"891","DOI":"10.1146\/annurev.immunol.22.012703.104543","article-title":"Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells","volume":"22","author":"Rot","year":"2004","journal-title":"Annu Rev Immunol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1124\/pr.113.04er14a","article-title":"International Union of Basic and Clinical Pharmacology. [corrected]. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors","volume":"66","author":"Bachelerie","year":"2014","journal-title":"Pharmacol Rev."},{"key":"B5","doi-asserted-by":"publisher","first-page":"2702","DOI":"10.1096\/fasebj.2.11.2840318","article-title":"Mechanism of neutrophil activation by NAF, a novel monocyte-derived peptide agonist","volume":"2","author":"Thelen","year":"1988","journal-title":"FASEB J."},{"key":"B6","doi-asserted-by":"publisher","first-page":"45","DOI":"10.1016\/j.jaut.2017.06.009","article-title":"Chemokine isoforms and processing in inflammation and immunity","volume":"85","author":"Proost","year":"2017","journal-title":"J Autoimmun."},{"key":"B7","doi-asserted-by":"publisher","first-page":"850","DOI":"10.1038\/ni.3201","article-title":"Dipeptidylpeptidase 4 inhibition enhances lymphocyte trafficking, improving both naturally occurring tumor immunity and immunotherapy","volume":"16","author":"Barreira da Silva","year":"2015","journal-title":"Nat Immunol."},{"key":"B8","doi-asserted-by":"publisher","first-page":"1895","DOI":"10.1073\/pnas.1716580115","article-title":"Accelerated wound healing in mice by on-site production and delivery of CXCL12 by transformed lactic acid bacteria","volume":"115","author":"V\u00e5gesj\u00f6","year":"2018","journal-title":"Proc Natl Acad Sci."},{"key":"B9","doi-asserted-by":"publisher","first-page":"1970","DOI":"10.3389\/fimmu.2017.01970","article-title":"Overview of the mechanisms that may contribute to the non-redundant activities of interferon-inducible CXC chemokine receptor 3 ligands","volume":"8","author":"Metzemaekers","year":"2017","journal-title":"Front Immunol."},{"key":"B10","doi-asserted-by":"publisher","first-page":"197","DOI":"10.1016\/j.pharmthera.2008.08.006","article-title":"Regulation of chemokine activity by posttranslational modification","volume":"120","author":"Mortier","year":"2008","journal-title":"Pharmacol Ther."},{"key":"B11","doi-asserted-by":"publisher","first-page":"483","DOI":"10.3389\/fimmu.2016.00483","article-title":"Regulation of chemokine activity - a focus on the role of dipeptidyl peptidase IV\/CD26","volume":"7","author":"Metzemaekers","year":"2016","journal-title":"Front Immunol."},{"key":"B12","doi-asserted-by":"publisher","first-page":"61","DOI":"10.1016\/j.cytogfr.2016.08.002","article-title":"The CXCL8-CXCR1\/2 pathways in cancer","volume":"31","author":"Liu","year":"2016","journal-title":"Cytokine Growth Factor Rev."},{"key":"B13","doi-asserted-by":"publisher","first-page":"593","DOI":"10.1586\/1744666X.2014.894886","article-title":"The CXCL8\/IL-8 chemokine family and its receptors in inflammatory diseases","volume":"10","author":"Russo","year":"2014","journal-title":"Expert Rev Clin Immunol."},{"key":"B14","doi-asserted-by":"publisher","first-page":"9233","DOI":"10.1073\/pnas.84.24.9233","article-title":"Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines","volume":"84","author":"Yoshimura","year":"1987","journal-title":"Proc Natl Acad Sci."},{"key":"B15","doi-asserted-by":"publisher","first-page":"1364","DOI":"10.1084\/jem.167.4.1364","article-title":"A novel, NH2-terminal sequence-characterized human monokine possessing neutrophil chemotactic, skin-reactive, and granulocytosis-promoting activity","volume":"167","author":"Van Damme","year":"1988","journal-title":"J Exp Med."},{"key":"B16","doi-asserted-by":"publisher","first-page":"883","DOI":"10.1016\/S0006-291X(88)80364-4","article-title":"Structure determination of a human lymphocyte derived neutrophil activating peptide (LYNAP)","volume":"151","author":"Gregory","year":"1988","journal-title":"Biochem Biophys Res Commun."},{"key":"B17","doi-asserted-by":"publisher","first-page":"755","DOI":"10.1016\/0006-291X(87)90432-3","article-title":"Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes","volume":"149","author":"Walz","year":"1987","journal-title":"Biochem Biophys Res Commun."},{"key":"B18","doi-asserted-by":"publisher","first-page":"285","DOI":"10.3389\/fimmu.2015.00285","article-title":"CXCL8 - the first chemokine","volume":"6","author":"Baggiolini","year":"2015","journal-title":"Front Immunol."},{"key":"B19","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1016\/0161-5890(89)90024-2","article-title":"Three forms of monocyte-derived neutrophil chemotactic factor (MDNCF) distinguished by different lengths of the amino-terminal sequence","volume":"26","author":"Yoshimura","year":"1989","journal-title":"Mol Immunol."},{"key":"B20","doi-asserted-by":"publisher","first-page":"2085","DOI":"10.1084\/jem.20080305","article-title":"Citrullination of CXCL8 by peptidylarginine deiminase alters receptor usage, prevents proteolysis, and dampens tissue inflammation","volume":"205","author":"Proost","year":"2008","journal-title":"J Exp Med."},{"key":"B21","doi-asserted-by":"publisher","first-page":"3768","DOI":"10.3390\/ijms19123768","article-title":"Differential effects of posttranslational modifications of CXCL8\/interleukin-8 on CXCR1 and CXCR2 internalization and signaling properties","volume":"19","author":"Vacchini","year":"2018","journal-title":"Int J Mol Sci."},{"key":"B22","doi-asserted-by":"publisher","first-page":"1167","DOI":"10.1002\/JLB.3AB0220-470R","article-title":"Truncation of CXCL8 to CXCL8(9-77) enhances actin polymerization and in vivo migration of neutrophils","volume":"107","author":"Metzemaekers","year":"2020","journal-title":"J Leukoc Biol."},{"key":"B23","doi-asserted-by":"publisher","first-page":"23128","DOI":"10.1016\/S0021-9258(18)54472-0","article-title":"Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities","volume":"266","author":"Clark-Lewis","year":"1991","journal-title":"J Biol Chem."},{"key":"B24","doi-asserted-by":"publisher","first-page":"2673","DOI":"10.1182\/blood.V96.8.2673","article-title":"Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades CTAP-III, PF-4, and GRO-alpha and leaves RANTES and MCP-2 intact","volume":"96","author":"Van den Steen","year":"2000","journal-title":"Blood."},{"key":"B25","doi-asserted-by":"publisher","first-page":"27348","DOI":"10.1074\/jbc.270.45.27348","article-title":"The functional role of the ELR motif in CXC chemokine-mediated angiogenesis","volume":"270","author":"Strieter","year":"1995","journal-title":"J Biol Chem."},{"key":"B26","doi-asserted-by":"publisher","first-page":"16075","DOI":"10.1016\/S0021-9258(17)33975-3","article-title":"Structural requirements for interleukin-8 function identified by design of analogs and CXC chemokine hybrids","volume":"269","author":"Clark-Lewis","year":"1994","journal-title":"J Biol Chem."},{"key":"B27","doi-asserted-by":"publisher","first-page":"e23913","DOI":"10.1371\/journal.pone.0023913","article-title":"Biological activity of CXCL8 forms generated by alternative cleavage of the signal peptide or by aminopeptidase-mediated truncation","volume":"6","author":"Mortier","year":"2011","journal-title":"PLoS ONE."},{"key":"B28","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1016\/S0076-6879(09)05401-9","article-title":"Chapter 1. Isolation, identification, and production of posttranslationally modified chemokines","volume":"461","author":"Loos","year":"2009","journal-title":"Methods Enzymol."},{"key":"B29","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1128\/AAC.12.1.11","article-title":"Human interferon: mass production in a newly established cell line, MG-63","volume":"12","author":"Billiau","year":"1977","journal-title":"Antimicrob Agents Chemother."},{"key":"B30","doi-asserted-by":"publisher","first-page":"1601","DOI":"10.1126\/science.2688092","article-title":"Endothelial interleukin-8: a novel inhibitor of leukocyte-endothelial interactions","volume":"246","author":"Gimbrone","year":"1989","journal-title":"Science."},{"key":"B31","doi-asserted-by":"crossref","first-page":"2223","DOI":"10.4049\/jimmunol.144.6.2223","article-title":"IL-1 alpha or tumor necrosis factor-alpha stimulate release of three NAP-1\/IL-8-related neutrophil chemotactic proteins in human dermal fibroblasts","volume":"144","author":"Schr\u00f6der","year":"1990","journal-title":"J Immunol."},{"key":"B32","doi-asserted-by":"crossref","first-page":"244","DOI":"10.4049\/jimmunol.142.1.244","article-title":"Secretion of novel and homologous neutrophil-activating peptides by LPS-stimulated human endothelial cells","volume":"142","author":"Schr\u00f6der","year":"1989","journal-title":"J Immunol."},{"key":"B33","doi-asserted-by":"crossref","first-page":"1000","DOI":"10.4049\/jimmunol.150.3.1000","article-title":"Identification of a novel granulocyte chemotactic protein (GCP-2) from human tumor cells. In vitro and in vivo comparison with natural forms of GRO, IP-10, and IL-8","volume":"150","author":"Proost","year":"1993","journal-title":"J Immunol."},{"key":"B34","doi-asserted-by":"publisher","first-page":"1189","DOI":"10.1002\/eji.1830190706","article-title":"The chemotactic activity for granulocytes produced by virally infected fibroblasts is identical to monocyte-derived interleukin 8","volume":"19","author":"Van Damme","year":"1989","journal-title":"Eur J Immunol."},{"key":"B35","doi-asserted-by":"publisher","first-page":"500","DOI":"10.1097\/BOR.0000000000000204","article-title":"Neutrophils in pediatric autoimmune disease","volume":"27","author":"Huttenlocher","year":"2015","journal-title":"Curr Opin Rheumatol."},{"key":"B36","doi-asserted-by":"publisher","first-page":"215","DOI":"10.1016\/j.molmed.2018.12.008","article-title":"Neutrophils in rheumatoid arthritis: breaking immune tolerance and fueling disease","volume":"25","author":"O'Neil","year":"2019","journal-title":"Trends Mol Med."},{"key":"B37","doi-asserted-by":"publisher","first-page":"589","DOI":"10.1136\/ard.2006.061853","article-title":"Blood and synovial fluid cytokine signatures in patients with juvenile idiopathic arthritis: a cross-sectional study","volume":"66","author":"de Jager","year":"2007","journal-title":"Ann Rheum Dis."},{"key":"B38","doi-asserted-by":"publisher","first-page":"13","DOI":"10.1186\/1546-0096-5-13","article-title":"Neutrophils: the forgotten cell in JIA disease pathogenesis","volume":"5","author":"Jarvis","year":"2007","journal-title":"Pediatr Rheumatol Online J."},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1002\/art.41605","article-title":"Synovial fluid neutrophils from patients with juvenile idiopathic arthritis display a hyperactivated phenotype","author":"Metzemaekers","year":"2021","journal-title":"Arthritis Rheumatol."},{"key":"B40","doi-asserted-by":"publisher","first-page":"471","DOI":"10.1016\/j.chom.2010.11.007","article-title":"Differential requirement for caspase-1 autoproteolysis in pathogen-induced cell death and cytokine processing","volume":"8","author":"Broz","year":"2010","journal-title":"Cell Host Microbe."},{"key":"B41","doi-asserted-by":"publisher","first-page":"266","DOI":"10.1038\/314266a0","article-title":"Homogeneous interferon-inducing 22K factor is related to endogenous pyrogen and interleukin-1","volume":"314","author":"Van Damme","year":"1985","journal-title":"Nature."},{"key":"B42","doi-asserted-by":"publisher","first-page":"273","DOI":"10.1172\/JCI1316","article-title":"Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies","volume":"101","author":"Schellekens","year":"1998","journal-title":"J Clin Invest."},{"key":"B43","doi-asserted-by":"publisher","first-page":"746","DOI":"10.1016\/j.molmed.2016.07.002","article-title":"Microbiomic and posttranslational modifications as preludes to autoimmune diseases","volume":"22","author":"Opdenakker","year":"2016","journal-title":"Trends Mol Med."},{"key":"B44","doi-asserted-by":"publisher","first-page":"4422","DOI":"10.1073\/pnas.0509158103","article-title":"Deimination of membrane-bound myelin basic protein in multiple sclerosis exposes an immunodominant epitope","volume":"103","author":"Musse","year":"2006","journal-title":"Proc Natl Acad Sci."},{"key":"B45","doi-asserted-by":"publisher","first-page":"614","DOI":"10.1007\/s13361-013-0822-7","article-title":"Quantification of intact and truncated stromal cell-derived factor-1alpha in circulation by immunoaffinity enrichment and tandem mass spectrometry","volume":"25","author":"Wang","year":"2014","journal-title":"J Am Soc Mass Spectrom."},{"key":"B46","doi-asserted-by":"publisher","first-page":"308","DOI":"10.1172\/JCI40594","article-title":"Evidence for an antagonist form of the chemokine CXCL10 in patients chronically infected with HCV","volume":"121","author":"Casrouge","year":"2011","journal-title":"J Clin Invest."},{"key":"B47","doi-asserted-by":"publisher","first-page":"545","DOI":"10.1002\/emmm.201100158","article-title":"The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions","volume":"3","author":"Cynis","year":"2011","journal-title":"EMBO Mol Med."},{"key":"B48","doi-asserted-by":"publisher","first-page":"235","DOI":"10.1021\/pr034086h","article-title":"Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA)","volume":"3","author":"Anderson","year":"2004","journal-title":"J Proteome Res."},{"key":"B49","doi-asserted-by":"publisher","first-page":"e1800361","DOI":"10.1002\/pmic.201800361","article-title":"Identification and quantification of proteoforms by mass spectrometry","volume":"19","author":"Schaffer","year":"2019","journal-title":"Proteomics."},{"key":"B50","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1039\/C9MO00154A","article-title":"High-throughput quantitative top-down proteomics","volume":"16","author":"Cupp-Sutton","year":"2020","journal-title":"Mol Omics."},{"key":"B51","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1146\/annurev-anchem-061516-045357","article-title":"Relative and absolute quantitation in mass spectrometry-based proteomics","volume":"11","author":"Ankney","year":"2018","journal-title":"Annu Rev Anal Chem."},{"key":"B52","doi-asserted-by":"publisher","first-page":"367","DOI":"10.1016\/j.it.2020.03.004","article-title":"Remnant epitopes generating autoimmunity: from model to useful paradigm","volume":"41","author":"Opdenakker","year":"2020","journal-title":"Trends Immunol."}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2021.644725\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,10,22]],"date-time":"2023-10-22T22:49:52Z","timestamp":1698014992000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2021.644725\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,3,11]]},"references-count":52,"alternative-id":["10.3389\/fimmu.2021.644725"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2021.644725","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,3,11]]},"article-number":"644725"}}