Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules

Abstract

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.

Rapid Online Oxidation of Proteins and Peptides via Electrospray-Accelerated Ozonation

“…The reagents utilized in these workflows and hence amino acid selectivity, reaction rates, and necessary considerations can vary widely. − ,,− ,− A concern of many labeling-based technologies is the influence derivatization has on the conformation of the protein. This is especially pertinent with these technologies, as many implementations generate multiply derivatized species and utilize reaction durations in the second to minute time scales.…”

DiLeu Isobaric Labeling Coupled with Limited Proteolysis Mass Spectrometry for High-Throughput Profiling of Protein Structural Changes in Alzheimer’s Disease

“…For example, top-down hydrogen/deuterium exchange (HDX) indicated that extensive deuteration takes place at the intervening loops and in the F-region, and fast photochemical oxidation of proteins (FPOPs) revealed that helix F has the higher accessibility among all apomyoglobin peptides . In addition, the fast fluoroalkylation of proteins (FFAPs) revealed that the increased modification of aromatic amino acids situated at helix F was observed in apomyoglobin . Overall, this leads us to conclude that the region helix F encompassing fully tryptic peptides, GHHEAELKPLAQSHATK, is readily accessible for PK and is more flexible in apomyoglobin.…”

“…Since fully tryptic peptide GLSDGEWQQVLNVWGK (2–17) is in helix A, together these findings indicate that helix A has solvent accessibility, in line with the phenomenon that has been documented by HDX, FPOP, and FFAP. For example, HDX revealed that helix A is only marginally developed with deuteration levels around 0.8, and the FPOP data showed that the protein segment (GLSDGEWQQVLNVWGK) (2–17) located on helix A gains different levels of protection with respect to folding time, and modification on helix A was also observed by FFAP . Overall, each method can provide insight into the unique features of surface accessibility and flexibility .…”

Utilization of Fast Photochemical Oxidation of Proteins and Both Bottom-up and Top-down Mass Spectrometry for Structural Characterization of a Transcription Factor–dsDNA Complex

“…To overcome this obstacle, hydroxyl radicals were introduced utilizing dissociation of water molecules by synchrotron irradiation . Because the availability of synchrotron for footprinting experiments is rather limited, alternative methods have been successfully tested to generate hydroxyl radicals based on the photolysis of hydrogen peroxide. − Later, other radicals were utilized as well to access the solvent-accessible surface area of proteins. − …”