{ "abstract": "Peptide mapping by liquid chromatography-tandem mass spectrometry (LC–MS/MS) is a key analytical strategy for residue-level characterization of antibody conjugates, providing modification-site assignments and apparent relative modification estimates that complement intact-mass and chromatographic drug-to-antibody ratio (DAR) measurements. The development of homogeneous antibody-drug conjugates (ADCs) relies on precise control of the conjugation site and DAR. For site-selectively modified ADCs, in which amino acid-level homogeneity is an important quality attribute, peptide mapping is therefore not only a confirmatory assay but also a central component of structural characterization. However, its reliability depends on enzymatic coverage and sample-handling conditions that affect the recovery of hydrophobic Fc-derived peptides, particularly in conjugates generated at high modification levels. In this study, we evaluated a multi-enzyme LC–MS/MS peptide-mapping workflow for antibody conjugates generated by microbial transglutaminase (MTG)-mediated Fc modification, using trastuzumab conjugated with dansylcadaverine (DNC) as a model system. An engineered thermostable MTG variant carrying five amino acid substitutions (D3C/S101P/G157S/G250R/G283C) was used to prepare a highly modified trastuzumab-DNC conjugate for evaluation of complementary protease coverage, residue-level modification-site assignment, and recovery of hydrophobic Fc-derived peptides. The engineered MTG was also compared with the commercial MTG formulation Activa using glycosylated trastuzumab under the specified nominal enzyme-loading conditions. Because this comparison was not normalized on the basis of catalytic activity under the antibody-conjugation conditions, it was treated as a formulation-based reference and was not used to establish intrinsic superiority of either enzyme preparation. Complementary digestion with trypsin, chymotrypsin, and Glu-C provided convergent evidence that DNC modification was predominantly localized to heavy-chain Q298 and enabled evaluation of other detectable glutamine-containing peptides. We further found that post-digestion addition of guanidine hydrochloride (GnHCl) during quenching increased the recovery of hydrophobic Fc-derived peptides, most notably the Q298-containing peptides. Omission of GnHCl during quenching reduced the recovery of several hydrophobic Fc-derived peptides and could bias peptide-map-based apparent modification estimates under the tested conditions. Under the optimized workflow, Q298 consistently showed the highest peptide-map-based apparent modification level, whereas apparent modification levels at other unambiguously assigned glutamine residues remained low under the tested conditions. However, these peak-area-based values do not represent absolute molar occupancy, and low-abundance off-site modifications below the analytical detection threshold cannot be excluded. These findings demonstrate that multi-enzyme peptide mapping combined with denaturant-assisted quenching provides a practical analytical workflow for residue-level characterization of highly modified, site-selective antibody conjugates. The workflow supports predominant modification at heavy-chain Q298 under the tested conditions and highlights the importance of sample-preparation optimization in peptide mapping-based characterization of ADCs and related antibody conjugates. Because DNC is a model primary amine, application of this workflow to therapeutic ADC linker-payloads will require substrate-specific optimization and analytical qualification.", "abstract_shown": true, "affiliations": [ "Ajinomoto Co., Inc. , 1-1 Suzuki-cho , , ," ], "article_type": null, "arxiv": null, "author_total": 7, "authors": [ { "affs": [ 0 ], "name": "Natsuki Shikida" }, { "affs": [ 0 ], "name": "Shunsuke Yamazaki", "orcid": "0009-0001-5507-8724" }, { "affs": [ 0 ], "name": "Tomohiro Watanabe", "orcid": "0009-0006-2242-1641" }, { "affs": [ 0 ], "name": "Haru Marumo" }, { "affs": [ 0 ], "name": "Kazutoshi Takahashi", "orcid": "0000-0003-3275-4109" }, { "affs": [ 0 ], "name": "Yutaka Matsuda", "orcid": "0000-0003-2801-4706" }, { "affs": [ 0 ], "name": "Kazutaka Shimbo" } ], "bibtex": "https://research.draconitic.com/paper/74f0wrt19jqnw.bib", "cited_by": 0, "doi": "10.1021/acsomega.6c04702", "fulltext_url": null, "id": "74f0wrt19jqnw", "issns": [ "2470-1343" ], "landing_url": "https://pubs.acs.org/acsodf/article/doi/10.1021/acsomega.6c04702/5453618/Multi-Enzyme-Peptide-Mapping-of-Site-Selective", "language": null, "licence": "CC-BY-NC-ND-4.0", "licence_label": "CC BY-NC-ND 4.0", "licence_source": "article", "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/", "metadata_licence": null, "native_subjects": [ [ "openalex", "2741|Monoclonal and Polyclonal Antibodies Research" ] ], "openalex": "W7222957610", "pdf_url": "https://pubs.acs.org/doi/pdf/10.1021/acsomega.6c04702", "pmcid": null, "pmid": null, "published": "2026-10-19", "publisher": "American Chemical Society (ACS)", "ris": "https://research.draconitic.com/paper/74f0wrt19jqnw.ris", "sources": [ { "first_seen": "2026-10-30T12:18:39Z", "native_id": "10.1021/acsomega.6c04702", "source": "crossref", "url": "https://pubs.acs.org/acsodf/article/doi/10.1021/acsomega.6c04702/5453618/Multi-Enzyme-Peptide-Mapping-of-Site-Selective" } ], "subjects": [ { "field": "medicine", "origin": "openalex:2741|Monoclonal and Polyclonal Antibodies Research", "subfield": "imaging" } ], "title": "Multi-Enzyme Peptide Mapping of Site-Selective Trastuzumab Modification by an Engineered Thermostable Microbial Transglutaminase", "title_text": "Multi-Enzyme Peptide Mapping of Site-Selective Trastuzumab Modification by an Engineered Thermostable Microbial Transglutaminase", "type": "peer-reviewed", "updated_at": "2026-10-30T12:18:39Z", "url": "https://research.draconitic.com/paper/74f0wrt19jqnw", "venue": "ACS Omega", "versions": [] }