Characterization of protein derivatives bearing a high degree of heterogeneity, such as immunoconjugates, results in a hard task. Middle approaches simplify MS-measurements, but it is difficult to correlate the degree of bioconjugation with changes in biological activity, owing to large fragments to which quantitative information is related. We developed a proteolysis method, named Domain Mapping (patent application IT2024000001524), which allows for recognition of single domains of protein structures. Thus, label-free quantification of bioconjugation could be related to peptide fragments in charge of structural and/or functional tasks. 

Trastuzumab was reacted with DOTA-NHS chelator in a kinetically controlled bioconjugation. The resulting immunoconjugate was reduced with DTT 5 mM and then alkylated with iodoacetamide 25mM in tris buffer pH 7,6 at 68°C. Afterwards, it was digested overnight at 37°C with trypsin, enzyme:antibody ratio 1:20, in tris buffer pH 7,6. Tryptic digest was analyzed through RP-LC-HRMS.

Limited proteolysis experiments were designed to obtain polypeptides as large as entire domains. We hypothesized that partial hot denaturation conditions could selectively unfold some domains, subsequently allowing trypsin to cut at the edges of the remaining folded. As a matter of fact, LC–MS analysis showed peptide fragments corresponding to whole undigested domains where intrachain disulfide bridges were not reduced, namely, FabHC (indicating the set of domains VH+CH1), CH3, VL and CH1. Coelution of conjugated and naked domain pairs allowed for relative MS¹ quantification of bioconjugation, while this latter was calculated for melted domains by differences with values measured in chain subunits analysis.

Domain mapping allowed for quantification of modified domains. Moreover, it might be useful for broader omics studies where proteolysis plays a key role, as well as a preparation method for single-domain antibodies.