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 cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, 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). FFAP uses fluoroalkyl radicals formed by reductive decomposition of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin-hemoglobin complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.

CF Plus Chemicals 62100 Brno Czech Republic

Department of Biochemistry Faculty of Science Charles University 12843 Prague Czech Republic

Institute of Biotechnology of the Czech Academy of Sciences 14220 Prague Czech Republic

Institute of Microbiology of the Czech Academy of Sciences 14220 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 16610 Prague Czech Republic

References provided by Crossref.org

Data-Independent Acquisition Represents a Promising Alternative for Fast Photochemical Oxidation of Proteins (FPOP) Samples Analysis

Structural Characterization of Monoclonal Antibodies and Epitope Mapping by FFAP Footprinting

An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans

Quantifying the Impact of the Peptide Identification Framework on the Results of Fast Photochemical Oxidation of Protein Analysis

Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Mass Spectrometry Analysis

Top-Down Proteoform Analysis by 2D MS with Quadrupolar Detection

Top-Down Detection of Oxidative Protein Footprinting by Collision-Induced Dissociation, Electron-Transfer Dissociation, and Electron-Capture Dissociation