Fast Fluoroalkylation of Proteins Uncovers Structure and Dynamics of Biological Macromolecules

Stable covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X Ray, NMR, or Cryo-EM used for protein structure determination. Although the current stable covalent labeling techniques enable to monitor protein solvent accessible areas with sufficient spatial resolution, there is still high demand for alternative, less complicated and inexpensive approaches. Here, we introduce a new covalent labeling method based on Fast Fluoroalkylation of Proteins (FFAP). The FFAP uses fluoroalkyl radicals formed by reductive decomposition of Togni reagents with ascorbic acid for labeling of proteins on a time scale of seconds. The feasibility of FFAP for effective labeling of proteins was demonstrated by monitoring differential amino acids modi-fication of native horse heart apomyoglobin/holomyoglobin and human haptoglobin-hemoglobin complex. The obtained data confirmed the Togni reagent-mediated FFAP as an advantageous alternative method for covalent labeling in applications, such as protein fingerprinting, epitope mapping and surface mapping of proteins (and their complexes) in general.