{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T05:16:57Z","timestamp":1788412617479,"version":"build-2803163510"},"reference-count":47,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Binex Co., Ltd."},{"id":[{"id":"https:\/\/ror.org\/00d806y18","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"crossref","award":["2022R1C1C1009883"],"award-info":[{"award-number":["2022R1C1C1009883"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"crossref","award":["RS-2025-16068462"],"award-info":[{"award-number":["RS-2025-16068462"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"crossref"}]},{"award":["2022R1C1C1009883"],"award-info":[{"award-number":["2022R1C1C1009883"]}],"id":[{"id":"https:\/\/ror.org\/013aysd81","id-type":"ROR","asserted-by":"publisher"}]},{"award":["RS-2025-16068462"],"award-info":[{"award-number":["RS-2025-16068462"]}],"id":[{"id":"https:\/\/ror.org\/013aysd81","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Glycosylation is a critical determinant of the efficacy, stability, and pharmacological behavior of therapeutic proteins. R27T, an engineered variant of interferon-\u03b21a, contains two N-glycosylation sites (Asn25 and Asn80), increasing its structural complexity and analytical requirements. In this study, we performed comprehensive total and site-specific glycan profiling of R27T using complementary analytical approaches. For total glycan analysis, the released N-glycans were fluorescently labeled with procainamide, providing enhanced sensitivity and broader glycan coverage compared with conventional 2-aminobenzamide labeling. Site-specific glycan profiling was performed by liquid chromatography\u2013tandem mass spectrometry (LC\u2013MS\/MS)-based peptide mapping. Protease digestion conditions were optimized to improve recovery of site-specific glycopeptides, with chymotrypsin identified as the most effective enzyme for resolving glycopeptides from individual glycosylation sites. Total glycan distributions reconstructed from peptide-mapping data were compared with fluorescence-based glycan profiling, showing that total and site-specific glycan data can be effectively combined. Minor discrepancies were observed depending on glycan structure, mainly due to differences in ionization efficiency. Distinct glycan distributions were observed between the two N-glycosylation sites of R27T. Molecular modeling further suggested that the additional glycan at Asn25 may enhance structural stability and receptor-binding affinity. These results demonstrate an integrative strategy for accurate glycan characterization in multi-site glycoproteins relevant to biotherapeutic development.<\/jats:p>","DOI":"10.3390\/ijms27177828","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T09:48:36Z","timestamp":1788256116000},"page":"7828","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Relative Quantitative Analysis of Site-Specific N-Linked Glycosylation in Hyperglycosylated Interferon-\u03b2 via Mass Spectrometry"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-7698-3612","authenticated-orcid":false,"given":"Daebong","family":"Moon","sequence":"first","affiliation":[{"name":"Binex Co., Ltd., Gaetbeol-ro 3, Yeonsu-gu, Incheon 21999, Republic of Korea"},{"name":"IGC Bioanalysis Center, Sondo Munhwa-ro 119, Incheon 21985, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"software","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]},{"given":"Geonwoo","family":"Kim","sequence":"additional","affiliation":[{"name":"Binex Co., Ltd., Gaetbeol-ro 3, Yeonsu-gu, Incheon 21999, Republic of Korea"},{"name":"IGC Bioanalysis Center, Sondo Munhwa-ro 119, Incheon 21985, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"software","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"}]},{"given":"Minjae","family":"Park","sequence":"additional","affiliation":[{"name":"Binex Co., Ltd., Gaetbeol-ro 3, Yeonsu-gu, Incheon 21999, Republic of Korea"},{"name":"IGC Bioanalysis Center, Sondo Munhwa-ro 119, Incheon 21985, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Bohyun","family":"Park","sequence":"additional","affiliation":[{"name":"Binex Co., Ltd., Gaetbeol-ro 3, Yeonsu-gu, Incheon 21999, Republic of Korea"},{"name":"IGC Bioanalysis Center, Sondo Munhwa-ro 119, Incheon 21985, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Na Young","family":"Kim","sequence":"additional","affiliation":[{"name":"R&D Center, ABION Inc., Seoul 08394, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"}]},{"given":"Woosung","family":"Son","sequence":"additional","affiliation":[{"name":"Novorex Inc., 302 Samhwan HIPEX A, 240 Pangyoyeok-ro, Bundang-gu, Seongnam-si 13493, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"visualization","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0896-718X","authenticated-orcid":false,"given":"Young Kee","family":"Shin","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Pathology and Cancer Genomics, Research Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea"},{"name":"Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul 08826, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3123-5745","authenticated-orcid":false,"given":"Kyoung","family":"Song","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Duksung Women\u2019s University, Seoul 01369, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Chen, B., Liu, W., Li, Y., Ma, B., Shang, S., and Tan, Z. (2022). Impact of N-linked glycosylation on therapeutic proteins. Molecules, 27.","DOI":"10.3390\/molecules27248859"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1223","DOI":"10.1002\/jps.21504","article-title":"Effects of glycosylation on the stability of protein pharmaceuticals","volume":"98","author":"Griebenow","year":"2009","journal-title":"J. Pharm. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1016\/j.ejps.2007.10.008","article-title":"pH Dependent effect of glycosylation on protein stability","volume":"33","author":"Wang","year":"2008","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1002\/1439-7633(20001117)1:4<214::AID-CBIC214>3.0.CO;2-B","article-title":"Glycopeptide synthesis and the effects of glycosylation on protein structure and activity","volume":"1","author":"Seitz","year":"2000","journal-title":"ChemBioChem"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"390","DOI":"10.1021\/jp203926r","article-title":"How hydrophobicity and the glycosylation site of glycans affect protein folding and stability: A molecular dynamics simulation","volume":"116","author":"Lu","year":"2011","journal-title":"J. Phys. Chem. B"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1626","DOI":"10.1002\/jps.20319","article-title":"Glycoengineering: The effect of glycosylation on the properties of therapeutic proteins","volume":"94","author":"Sinclair","year":"2005","journal-title":"J. Pharm. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Mastrangeli, R., Satwekar, A., and Bierau, H. (2023). Innovative metrics for reporting and comparing the glycan structural profile in biotherapeutics. Molecules, 28.","DOI":"10.3390\/molecules28083304"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1","DOI":"10.18103\/mra.v10i3.2715","article-title":"Biobetters: Are they truly better?","volume":"10","author":"Zaveri","year":"2022","journal-title":"Med. Res. Arch."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1021\/acs.analchem.6b04343","article-title":"Recent Advances in the Analysis of Complex Glycoproteins","volume":"89","author":"Gaunitz","year":"2017","journal-title":"Anal. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"102263","DOI":"10.1016\/j.cbpa.2023.102263","article-title":"Recent advances on the synthesis of N-linked glycoprotein for the elucidation of glycan functions","volume":"73","author":"Liu","year":"2023","journal-title":"Curr. Opin. Chem. Biol."},{"key":"ref_11","first-page":"13","article-title":"The sweet spot for biologics: Recent advances in characterization of biotherapeutic glycoproteins","volume":"15","author":"Greville","year":"2017","journal-title":"Expert Rev. Proteom."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.aca.2016.03.049","article-title":"Glycan characterization of biopharmaceuticals: Updates and perspectives","volume":"921","author":"Planinc","year":"2016","journal-title":"Anal. Chim. Acta"},{"key":"ref_13","first-page":"S17","article-title":"Interferon-\u03b2 mechanisms of action in multiple sclerosis","volume":"74","author":"Marks","year":"2010","journal-title":"Neurology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"682","DOI":"10.1212\/WNL.51.3.682","article-title":"Interferon beta in the treatment of multiple sclerosis","volume":"51","author":"Chabot","year":"1998","journal-title":"Neurology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"13957","DOI":"10.18632\/oncotarget.14573","article-title":"Sensitization of glycoengineered interferon-\u03b21a-resistant cancer cells by Cflip inhibition for enhanced anti-cancer therapy","volume":"8","author":"Kim","year":"2017","journal-title":"Oncotarget"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1203","DOI":"10.1007\/s000180050248","article-title":"The structure of human interferon-beta: Implications for activity","volume":"54","author":"Karpusas","year":"1998","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"563","DOI":"10.2165\/00023210-200216080-00005","article-title":"Comparative assessment of immunomodulating therapies for relapsing-remitting multiple sclerosis","volume":"16","author":"Khan","year":"2002","journal-title":"CNS Drugs"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"358","DOI":"10.1212\/WNL.49.2.358","article-title":"Impact of interferon beta-1a on neurologic disability in relapsing multiple sclerosis","volume":"49","author":"Rudick","year":"1997","journal-title":"Neurology"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"8003","DOI":"10.1074\/jbc.273.14.8003","article-title":"Differences in activity between \u03b1 and \u03b2 type I interferons explored by mutational analysis","volume":"273","author":"Runkel","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.9734\/BJAST\/2016\/29679","article-title":"Impact of glycosylation on therapeutic glycoproteins","volume":"18","author":"Mousavi","year":"2016","journal-title":"Br. J. Appl. Sci. Technol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"8412","DOI":"10.1038\/s41598-020-65495-y","article-title":"Stability and activity of the hyperglycosylated human interferon-\u03b2 R27T variant","volume":"10","author":"Song","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"6619","DOI":"10.1021\/acsomega.9b04385","article-title":"Glycosylation heterogeneity of hyperglycosylated recombinant human interferon-\u03b2 (rhIFN-\u03b2)","volume":"5","author":"Song","year":"2020","journal-title":"ACS Omega"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1021\/acs.analchem.9b04651","article-title":"Recent advances in glycoproteomic analysis by mass spectrometry","volume":"92","author":"Suttapitugsakul","year":"2020","journal-title":"Anal. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1007\/s10719-012-9443-9","article-title":"Strategies for the profiling, characterisation and detailed structural analysis of N-linked oligosaccharides","volume":"30","author":"Tharmalingam","year":"2013","journal-title":"Glycoconj. J."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Lee, S., Son, W.S., Yang, H.B., Rajasekaran, N., Kim, S.-S., Hong, S., Choi, J.-S., Choi, J.Y., Song, K., and Shin, Y.K. (2019). A glycoengineered interferon-\u03b2 mutein (R27T) generates prolonged signaling by an altered receptor-binding kinetics. Front. Pharmacol., 9.","DOI":"10.3389\/fphar.2018.01568"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Song, K., Yoon, I.-S., Kim, N.A., Kim, D.-H., Lee, J., Lee, H.J., Lee, S., Choi, S., Choi, M.-K., and Kim, H.H. (2014). Glycoengineering of interferon-\u03b2 1a improves its biophysical and pharmacokinetic properties. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0096967"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3457","DOI":"10.1007\/s00216-010-3532-z","article-title":"Glycan labeling strategies and their use in identification and quantification","volume":"397","author":"Ruhaak","year":"2010","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1007\/s00216-010-3988-x","article-title":"HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals","volume":"398","author":"Melmer","year":"2010","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1038\/nmeth808","article-title":"Versatile fluorescent derivatization of glycans for glycomic analysis","volume":"2","author":"Xia","year":"2005","journal-title":"Nat. Methods"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1002\/1615-9861(200102)1:2<311::AID-PROT311>3.0.CO;2-J","article-title":"Identification of protein-bound carbohydrates by mass spectrometry","volume":"1","author":"Harvey","year":"2001","journal-title":"Proteomics"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/j.ejps.2015.07.020","article-title":"Basal buffer systems for a newly glycosylated recombinant human interferon-\u03b2 with Biophysical stability and DOE approaches","volume":"78","author":"Kim","year":"2015","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1007\/s13361-014-0998-5","article-title":"On the ionization and ion transmission efficiencies of different ESI-MS interfaces","volume":"26","author":"Cox","year":"2014","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.chroma.2016.06.085","article-title":"High throughput peptide mapping method for analysis of site specific monoclonal antibody oxidation","volume":"1460","author":"Li","year":"2016","journal-title":"J. Chromatogr. A"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"4175","DOI":"10.1021\/ac0602266","article-title":"Enhancing electrospray ionization efficiency of peptides by derivatization","volume":"78","author":"Mirzaei","year":"2006","journal-title":"Anal. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2111","DOI":"10.1007\/s00216-018-0848-6","article-title":"Development of an LC-MS\/MS peptide mapping protocol for the NISTmab","volume":"410","author":"Mouchahoir","year":"2018","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1571","DOI":"10.1021\/acs.jproteome.3c00633","article-title":"An XIC-centric strategy for improved identification and quantification in proteomic data analyses","volume":"23","author":"Wang","year":"2024","journal-title":"J. Proteome Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"37189","DOI":"10.1038\/srep37189","article-title":"Classification of Tandem Mass Spectra for Identification of N- and O-linked Glycopeptides","volume":"6","author":"Eshghi","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1097\/IM9.0000000000000071","article-title":"Mass spectrometry analysis of SARS-CoV-2 nucleocapsid protein reveals camouflaging glycans and unique post-translational modifications","volume":"3","author":"Sun","year":"2021","journal-title":"Infect. Microbes Dis."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/978-1-0716-1241-5_1","article-title":"Glycosylation of Therapeutic Proteins: A Critical Quality Attribute","volume":"2271","author":"Delobel","year":"2021","journal-title":"Mass Spectrom. Glycoproteins"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1016\/j.drudis.2018.01.009","article-title":"On the glycosylation aspects of biosimilarity","volume":"23","author":"Hajba","year":"2018","journal-title":"Drug Discov. Today"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1299","DOI":"10.1038\/nprot.2012.063","article-title":"Structural analysis of N- and O-glycans released from glycoproteins","volume":"7","author":"Jensen","year":"2012","journal-title":"Nat. Protoc."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1016\/j.sbi.2009.05.005","article-title":"Mass spectrometry in the analysis of N-linked and O-linked glycans","volume":"19","author":"North","year":"2009","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_43","unstructured":"Protein Metrics (2025, December 05). Quantification of Modifications in the Peptide Workflows. Available online: https:\/\/support.proteinmetrics.com\/hc\/en-us\/articles\/20700549870612-Quantification-of-Modifications-in-the-Peptide-Workflows."},{"key":"ref_44","first-page":"107","article-title":"Estimation and analysis methods for trastuzumab deamidation levels using mass spectrometry","volume":"15","author":"Moon","year":"2024","journal-title":"Mass Spectrom. Lett."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1093\/glycob\/cwaf048","article-title":"Modeling glycans with AlphaFold 3: Capabilities, caveats, and limitations","volume":"35","author":"Huang","year":"2025","journal-title":"Glycobiology"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1135","DOI":"10.1093\/bioinformatics\/btz740","article-title":"EvoEF2: Accurate and fast energy function for computational protein design","volume":"36","author":"Huang","year":"2019","journal-title":"Bioinformatics"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/17\/7828\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T04:41:01Z","timestamp":1788410461000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/17\/7828"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,1]]},"references-count":47,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["ijms27177828"],"URL":"https:\/\/doi.org\/10.3390\/ijms27177828","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,1]]}}}