{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T04:38:27Z","timestamp":1779338307512,"version":"3.51.4"},"reference-count":29,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2018,12,5]],"date-time":"2018-12-05T00:00:00Z","timestamp":1543968000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2018,12,5]],"date-time":"2018-12-05T00:00:00Z","timestamp":1543968000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Conjugation of small molecules to proteins through N-hydroxysuccinimide (NHS) esters results in a random distribution of small molecules on lysine residues and the protein N-terminus. While mass spectrometry methods have improved characterization of these protein conjugates, it remains a challenge to quantify the occupancy at individual sites of conjugation. Here, we present a method using Tandem Mass Tags (TMT) that enabled the accurate and sensitive quantification of occupancy at individual conjugation sites in the NIST monoclonal antibody. At conjugation levels relevant to antibody drug conjugates in the clinic, site occupancy data was obtained for 37 individual sites, with average site occupancy data across 2 adjacent lysines obtained for an additional 12 sites. Thus, altogether, a measure of site occupancy was obtained for 98% of the available primary amines. We further showed that removal of the Fc-glycan on the NIST mAb increased conjugation at two specific sites in the heavy chain, demonstrating the utility of this method to identify changes in the susceptibility of individual sites to conjugation. This improved site occupancy data allowed calibration of a bi-parametric linear model for predicting the susceptibility of individual lysines to conjugation from 3D-structure based on their solvent exposures and ionization constants. Trained against the experimental data for lysines from the Fab fragment, the model provided accurate predictions of occupancies at lysine sites from the Fc region and the protein N-terminus (R<jats:sup>2<\/jats:sup>\u2009=\u20090.76). This predictive model will enable improved engineering of antibodies for optimal labeling with fluorophores, toxins, or crosslinkers.<\/jats:p>","DOI":"10.1038\/s41598-018-35924-0","type":"journal-article","created":{"date-parts":[[2018,11,29]],"date-time":"2018-11-29T18:39:18Z","timestamp":1543516758000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["An accurate TMT-based approach to quantify and model lysine susceptibility to conjugation via N-hydroxysuccinimide esters in a monoclonal antibody"],"prefix":"10.1038","volume":"8","author":[{"given":"Jennifer J.","family":"Hill","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tammy-Lynn","family":"Tremblay","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christopher R.","family":"Corbeil","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Enrico O.","family":"Purisima","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Traian","family":"Sulea","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2018,12,5]]},"reference":[{"key":"35924_CR1","doi-asserted-by":"publisher","first-page":"3863","DOI":"10.1021\/acs.biochem.7b00536","volume":"56","author":"JN deGruyter","year":"2017","unstructured":"deGruyter, J. N., Malins, L. R. & Baran, P. S. Residue-Specific Peptide Modification: A Chemist\u2019s Guide. Biochemistry 56, 3863\u20133873, https:\/\/doi.org\/10.1021\/acs.biochem.7b00536 (2017).","journal-title":"Biochemistry"},{"key":"35924_CR2","doi-asserted-by":"publisher","first-page":"315","DOI":"10.1038\/nrd.2016.268","volume":"16","author":"A Beck","year":"2017","unstructured":"Beck, A., Goetsch, L., Dumontet, C. & Corvaia, N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat. Rev. Drug Discov. 16, 315\u2013337, https:\/\/doi.org\/10.1038\/nrd.2016.268 (2017).","journal-title":"Nat. Rev. Drug Discov."},{"key":"35924_CR3","doi-asserted-by":"publisher","first-page":"469","DOI":"10.2174\/1568009616666160512144715","volume":"16","author":"W Gao","year":"2016","unstructured":"Gao, W. et al. Recent Advances in Site Specific Conjugations of Antibody Drug Conjugates (ADCs). Curr. Cancer Drug Targets 16, 469\u2013479 (2016).","journal-title":"Curr. 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