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. 2026 Jan 24;37(2):281–292. doi: 10.1021/acs.bioconjchem.5c00519

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Mass spectrometry analysis of covalent conjugate between the NanoBondy and CD45d. (A) Dominant cross-linking sites. 2H5 R72C anti-CD45 NanoBondy was incubated with CD45d before cross-linking MS/MS. The number of identified cross-linked spectral matches for each NanoBondy cross-linking site on CD45 is shown. (B) Higher energy collision-induced dissociation (HCD) fragmentation spectrum of identified cross-link precursor ions corresponding to D173 (NanoBondy) to K131 (CD45d). Fragment ions matching fragmented cross-link (with cross-linker still intact) are annotated in bold, while peaks corresponding to fragments post-cross-link fragmentation are annotated with nonbold lines. “Car” indicates carbamidomethylation of cysteine. (C) Mapping of cross-link sites. AlphaFold prediction of NanoBondy (purple) bound to CD45d1d2 domains (green), highlighting cross-linking sites identified from the reactive aspartate (cyan) of the NanoBondy to target lysines (pink) or to serine (dark blue). Samples were run in technical triplicate. The cross-linking MS experiment was conducted twice.