Fast FluoroAlkylation of Proteins (FFAP) as a new tool for characterization of protein therapeutics

Covalent labeling combined with mass spectrometry is a robust method for studying protein structure, interactions, and dynamics. Recently, a new technique called Fast FluoroAlkylation of Proteins (FFAP) has expanded the toolbox of covalent labeling techniques. FFAP is a novel protein labeling method that uses fluoroalkyl radicals generated from hypervalent Togni reagents. These radicals target aromatic aminoacids and cystein and are beneficial for structural characterization of monoclonal antibodies (and biosimilars) as well as epitope mapping.

The Togni reagents are decomposed by ascorbic acid to form fluoroalkyl radicals, which then label the solvent-accessible aromatic amino acid side chains of both Trastuzumab and the human HER2 receptor, alone and in complex, for a short duration using a quench-flow microcapillary apparatus. Following this, Trastuzumab and the HER2 receptor are reduced, alkylated, and digested by trypsin/Lys-C. The resulting peptides are separated on a reversed-phase column (Luna Omega Polar C18, 100Å, Phenomenex) in a water-acetonitrile gradient (Agilent 1290, Agilent Technologies) and analyzed by high-resolution mass spectrometry (solariX XR 15T, Bruker Daltonics). The extent of modification is determined from the intensity of the first isotope at the peak maximum using DataAnalysis software (Bruker Daltonics).

Initially, different batches of Trastuzumab (a monoclonal anti-HER2 antibody marketed as Herceptin) were labeled with fluoroalkyl radicals, revealing consistent solvent-accessible regions in both batches, particularly in the Fab fragment including the antigen-binding sites. These results align with the X-ray structural model (1N8Z). Further testing of our novel fluoroalkyl radical technology for epitope mapping on the Trastuzumab-HER2 complex identified differences in the modification of free proteins and the complex, uncovering residues at the interaction interface that correspond with previous structural analyses. Additionally, the data revealed a minor structural rearrangement of the HER2 receptor upon Trastuzumab binding, explained by the remodeling of the β-sheet structure into an unstructured loop, a behavior previously observed in rat HER2 (1N8Y) and the human HER2 receptor–Pertuzumab complex.

This work was mainly supported by the Czech Science Foundation (22-27695S), NPO-NEURO-EXCELLES (LX22NPO5107) and by European Commission H2020 (EPIC-XS grant agreement ID: 823839; EU FT-ICR MS grant agreement ID: 731077), and the Grant Agency of Charles University (grant number 359221) and, in part, by the Czech Academy of Sciences (RVO61388971). We also acknowledge the Structural Mass Spectrometry Core Facility of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2018127) and European Regional Development Fund Project "UP CIISB" (No. CZ.02.1.01/0.0/0.0/18_046/ 0015974).