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However, the technique is still rarely coupled to separation techniques, as the widely used reversed\u2010phase chromatography mostly denatures proteins. Here we present a study combining both capillary zone electrophoresis and size exclusion chromatography, each coupled online to native electrospray ionization mass spectrometry for the analysis of proteoforms of plasminogen. Plasminogen, the zymogen form of plasmin, is an abundant plasma protein with multiple proteoforms whose activation via proteolytic cleavage is tightly regulated within the fibrinolytic system and is partly modulated by post\u2010translational modifications. Near\u2010native CZE conditions enable the separation of proteoforms differing in phosphorylation and glycosylation, that is, almost baseline separation of proteoforms differing in phosphorylation and sialylation. The nanoflow sheath liquid interface (nanoCEasy) enables efficient ionization as well as the flexibility to ionize under native or denaturing conditions. The CZE\u2010MS set\u2010up can be used to study proteoform\u2010resolved interaction directly in the capillary, as demonstrated for the activation of plasminogen by the co\u2010injection of tissue plasminogen activator (tPA). In these proof\u2010of\u2010concept experiments, various truncations of plasminogen were observed with a preferred cleavage for the N\u2010glycosylated and the phosphorylated proteoforms. SEC\u2010MS enables partial separation of the proteoforms, particularly between glycosylated and nonglycosylated proteoforms leading to the detection of new proteoforms not described by direct infusion experiments. Overall, the combination of near\u2010native CZE\u2010MS and SEC\u2010MS provides a detailed characterization of plasminogen and enables proteoforms\u2010resolved interaction studies.<\/jats:p>","DOI":"10.1002\/jms.70048","type":"journal-article","created":{"date-parts":[[2026,3,17]],"date-time":"2026-03-17T00:10:02Z","timestamp":1773706202000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Proteoform\u2010Resolved Interaction Studies of Plasminogen by CZE\u2010MS and SEC\u2010MS Under Near\u2010Native Conditions"],"prefix":"10.1002","volume":"61","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2404-4924","authenticated-orcid":false,"given":"Christian","family":"Neus\u00fc\u00df","sequence":"first","affiliation":[{"name":"Aalen University  Aalen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hadi","family":"Lioe","sequence":"additional","affiliation":[{"name":"CSL  Melbourne Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toby","family":"Dite","sequence":"additional","affiliation":[{"name":"CSL  Melbourne Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-6414-7385","authenticated-orcid":false,"given":"Sawyen","family":"Ow","sequence":"additional","affiliation":[{"name":"CSL  Melbourne Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthias","family":"Pelzing","sequence":"additional","affiliation":[{"name":"CSL  Melbourne Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,3,16]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1265"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1002\/jms.5095"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2025.102993"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.accounts.2c00216"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.1c00212"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.1c00293"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1098-2787(1997)16:1<1::AID-MAS1>3.0.CO;2-L"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.talanta.2024.126824"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jpba.2020.113207"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jproteome.4c00430"},{"key":"e_1_2_7_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.aca.2023.341360"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jchromb.2017.05.010"},{"key":"e_1_2_7_14_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.8b03333"},{"key":"e_1_2_7_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jchromb.2018.04.010"},{"key":"e_1_2_7_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jchromb.2017.09.027"},{"key":"e_1_2_7_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijms.2020.116502"},{"key":"e_1_2_7_18_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac502590h"},{"key":"e_1_2_7_19_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-2493-7"},{"key":"e_1_2_7_20_1","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.70012"},{"key":"e_1_2_7_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/elps.202000317"},{"key":"e_1_2_7_22_1","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.202300135"},{"key":"e_1_2_7_23_1","doi-asserted-by":"publisher","DOI":"10.1007\/s13361-017-1781-1"},{"key":"e_1_2_7_24_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202408370"},{"key":"e_1_2_7_25_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.5c02760"},{"key":"e_1_2_7_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.aca.2024.343287"},{"key":"e_1_2_7_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijms.2021.116541"},{"key":"e_1_2_7_28_1","doi-asserted-by":"publisher","DOI":"10.1002\/elps.201800067"},{"key":"e_1_2_7_29_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.0c04975"},{"key":"e_1_2_7_30_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.1c03560"},{"key":"e_1_2_7_31_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-47772-w"},{"key":"e_1_2_7_32_1","volume-title":"Capillary Electrophoresis\u2010Mass Spectrometry for Proteomics and Metabolomics. 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