Carbocation Footprinting of Soluble and Transmembrane Proteins

Abstract

Here, we introduce carbocations (R 3 C + ) as laser-initiated footprinting reagents for proteins. We screened seven candidates and selected trifluomethoxy benzyl bromide (TFBB) as an effective precursor for the electrophilic trifluomethoxy benzyl carbocation (TFB + ) under laser (248 nm) irradiation on the fast photochemical oxidation of proteins (FPOP) platform. Initial results demonstrate that this electrophilic cation reagent affords residue coverage of nucleophilic amino acids including H, W, M, and S. Further, the addition of TFB + increases the hydrophobicity of the peptides so that separation of isomeric peptide products by reversed-phase LC is improved, suggesting opportunities for subresidue footprinting. Comparison of apo-and holo-myoglobin footprints shows that the TFB + footprinting is sensitive to protein conformational change and solvent accessibility. Interestingly, because the TFB + is amphiphilic, the reagent can potentially footprint membrane proteins as demonstrated for vitamin K epoxide reductase (VKOR) stabilized in a micelle. Not only does footprinting of the extra-membrane domain occur, but also some footprinting of the hydrophobic transmembrane domain is achieved owing to the interaction of TFB + with the micelle. Carbocation precursors are stable and amenable for tailoring their properties and those of the incipient carbocation, enabling targeting their soluble or membrane-associated or embedded regions and distinguishing between the extra-and trans-membrane domains of membrane proteins.

Benzoyl Transfer for Footprinting Alcohol-Containing Residues in Higher Order Structural Applications of Mass-Spectrometry-Based Proteomics

“…These findings launch BF as a novel footprinter for application to other S-, T-, Y-rich binding sites, most notably antibody paratopes. BF affords broad reactivity also observed for stronger nucleophiles: protein N-term, K, and H. Furthermore, acyl transfer reagents offer opportunities in the future to add substituents to the aromatic ring and tailor the reactivity or hydrophobicity for footprinting membrane-bound proteins . Overall, the S-, T-, Y-reactivities reported here and the tunability of the BF solubility by substitution of the aryl ring should endow BF footprinting the opportunity to modify critical alcoholic residues in a variety of environments.…”

Advances in mass spectrometry‐based footprinting of membrane proteins

“…Carbene diradicals are being developed for protein footprinting, but mostly for soluble proteins [22,23,[81][82][83][84]. however, found that the photoactivatable aryl diazirines (Figure 4B) Recently, Sun et al [85] introduced a new class of reactive species for protein footprinting, namely carbocations. Carbocations are highly reactive intermediates with lifetimes as short as nanoseconds [86].…”

“…Recently, Sun et al. [85] introduced a new class of reactive species for protein footprinting, namely carbocations. Carbocations are highly reactive intermediates with lifetimes as short as nanoseconds [86].…”

Investigating Daptomycin–Membrane Interactions Using Native MS and Fast Photochemical Oxidation of Peptides in Nanodiscs

“…Oxidative modifications to these species result in predictable mass shifts that are detected by MS. FPOP has been used to investigate a variety of proteins with model membrane systems such as nanodiscs, , micelles, bicelles, and even living organisms . Other rapid footprinting chemistries have also been successfully used for membrane proteins, including the trifluoromethyl radical (·CF 3 ), carbenes derived from diazirines, and carbocations. − Slower covalent labeling reagents, including glycine ethyl ester diethylpyrocarbonate have also proven effective. The field is rapidly advancing with the development of new reagents that differ in reactivities, kinetics, and selectivity to enhance structural investigations of diverse targets.…”