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.
Publication Date: 2021-11-30 PubMed ID: 34846870DOI: 10.1021/jacs.1c07771Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research introduces a novel method for covalent labeling of proteins, called Fast Fluoroalkylation of Proteins (FFAP), which offers a quick, efficient, and less complicated alternative to the existing techniques in protein structure determination.
Understanding Covalent Labeling and Need for FFAP
- Covalent labeling of proteins, used in combination with mass spectrometry, is a technique utilized in determining the structure of biological macromolecules. Typically, these methods are used alongside traditional structural methods such as X-ray crystallography, NMR spectroscopy, and Cryo-EM.
- While current covalent labeling techniques are able to monitor solvent-accessible regions on proteins with adequate spatial resolution, there’s a requirement for less complicated, cost-efficient, and quicker alternative techniques. This forms the basis for the introduction of Fast Fluoroalkylation of Proteins (FFAP).
Fast Fluoroalkylation of Proteins (FFAP)
- The new labeling method, FFAP, uses fluoroalkyl radicals. These are generated by the reductive decomposition of Togni reagents with ascorbic acid and are deployed for labeling proteins on a time scale of seconds. The speed of this method is an advantage against traditional methods.
- Two proteins – horse heart apomyoglobin/holomyoglobin and the human haptoglobin-hemoglobin complex – were used to demonstrate FFAP’s effectiveness. The researchers monitored differential amino acids modification for these proteins, proving the feasibility of the FFAP method.
Applications and Access to the Research Data
- One of the beneficial applications of FFAP is for protein footprinting and epitope mapping of proteins and their complexes. Any sector that needs efficient and effective protein characterization could potentially benefit from this method.
- Furthemore, in the spirit of aiding scientific study and creating a more transparent research environment, data collected from this research is available to the public via the ProteomeXchange server with the data set identifier PXD027310.
Cite This Article
APA
Fojtík L, Fiala J, Pompach P, Chmelík J, Matoušek V, Beier P, Kukačka Z, Novák P.
(2021).
Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules.
J Am Chem Soc, 143(49), 20670-20679.
https://doi.org/10.1021/jacs.1c07771 Publication
Researcher Affiliations
- Institute of Microbiology of the Czech Academy of Sciences, 14220 Prague, Czech Republic.
- Department of Biochemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic.
- Institute of Microbiology of the Czech Academy of Sciences, 14220 Prague, Czech Republic.
- Department of Biochemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic.
- Institute of Microbiology of the Czech Academy of Sciences, 14220 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.
- Department of Biochemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic.
- CF Plus Chemicals, 62100 Brno, Czech Republic.
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 16610 Prague, Czech Republic.
- Institute of Microbiology of the Czech Academy of Sciences, 14220 Prague, Czech Republic.
- Institute of Microbiology of the Czech Academy of Sciences, 14220 Prague, Czech Republic.
MeSH Terms
- Alkylation
- Animals
- Escherichia coli / chemistry
- Escherichia coli Proteins / chemistry
- Haptoglobins / chemistry
- Hemoglobins / chemistry
- Horses
- Humans
- Hydrocarbons, Fluorinated / chemistry
- Mass Spectrometry / methods
- Myoglobin / chemistry
- Protein Conformation
- Repressor Proteins / chemistry
Citations
This article has been cited 16 times.- Lu H, Wang B, Liu Y, Wang D, Fields L, Zhang H, Li M, Shi X, Zetterberg H, Li L. DiLeu Isobaric Labeling Coupled with Limited Proteolysis Mass Spectrometry for High-Throughput Profiling of Protein Structural Changes in Alzheimer's Disease. Anal Chem 2023 Jul 4;95(26):9746-9753.
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