Scavenging with TEMPO^• To Identify Peptide- and Protein-Based Radicals by Mass Spectrometry:  Advantages of Spin Scavenging over Spin Trapping

Abstract

The detection and characterization of radicals in biomolecules are challenging due to their high reactivity and low concentration. Mass spectrometry (MS) provides a tool for the unambiguous identification of protein-based radicals by exploiting their reactivity with suitable reagents. To date, protein-radical detection by MS has been modeled after electron paramagnetic resonance experiments, in which diamagnetic spin traps, such as 3,5-dibromo-4-nitrosobenzene sulfonic acid, convert unstable radicals to more stable spin adducts. Since MS detects mass changes, and not unpaired spins, conversion of radicals to stable diamagnetic adducts is more desirable. The use of 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO(*)) in the MS identification of protein-based radicals was explored here to establish whether scavenging via radical combination would give rise to TEMPO adducts that were stable to MS analysis. The horseradish peroxidase/H(2)O(2) reaction was used to generate radicals in derivatives of tyrosine, tryptophan, and phenylalanine as models of protein-based radicals. TEMPO(*) was added as a radical scavenger, and the products were analyzed by electrospray ionization (ESI) MS. Dramatically higher mass-adduct yields were obtained using radical scavenging vs radical trapping, which greatly enhanced the sensitivity of radical detection. The efficiency of TEMPO(*) in protein radical scavenging was examined in horse heart myoglobin and cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae. On H(2)O(2) binding to their ferric hemes, two oxidizing equivalents are transferred to the proteins as an Fe(IV)=O species and a polypeptide-based radical. In addition, CCP has been shown to reduce up to 10 equiv of H(2)O(2) using endogenous donors, thereby generating as many as 20 radicals on its polypeptide. Following myoglobin and CCP incubation with a 10-fold molar excess of H(2)O(2) and TEMPO(*), matrix-assisted laser desorption ionization (MALDI) time-of-flight analysis of the tryptic peptides derived from the proteins revealed 1 and 9 TEMPO adducts of myoglobin and CCP, respectively. Given the high scavenging efficiency of TEMPO(*) and the stability of TEMPO-labeled peptides in ESI and MALDI sources, scavenging by stable nitroxide radicals coupled with MS analysis should provide sensitive and powerful technology for the characterization of protein-based radicals.

Inhibition of myeloperoxidase-mediated protein nitration by tempol: Kinetics, mechanism, and implications

“…2) that does not coincide with tempol spectrum lines. Thus, micromolar tempol competes with millimolar DBNBS for the RNase-tyr • , a fact that is in agreement with the known second-order rate constants of nitroxides and DBNBS reacting with carboncentered radicals (22). In line with the lower HRP efficiency in nitrating RNase compared with MPO ( Fig.…”

“…One possibility to explain tempol consumption was recombination reactions with RNase-tyr • (Eq. 2) (7,22). To test this possibility, we examined radical production by EPR spin-trapping experiments with 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS).…”

Identification of Free Radicals on Hemoglobin from its Self-peroxidation Using Mass Spectrometry and Immuno-spin Trapping

“…The mass of the b 2 ion corresponds to amino acids threonine plus a DMPO-modified tyrosine. In addition, the observation of the y 14 and y 15 ions provides the necessary data to definitively assign the location of the DMPO adduct. The observed mass difference between the y 14 and y 15 ions corresponds to the mass of a tyrosine residue plus the mass of one DMPO molecule.…”

“…These methods, however, suffer from potential drawbacks, such as the protein's stability and conformation can be perturbed by mutations and the formation of the amino acid radical and transfer of the radical is expected to be affected. Recently, mass spectrometry has been utilized to study protein free radicals (11)(12)(13)(14)(15). The spin trap molecules form a covalent bond with the protein resulting in a new nitroxide radical, which may be stable under electrospray ionization mass spectrometry conditions.…”

Experimental evidence for competitive N O and O C bond homolysis in gas-phase alkoxyamines

“…[38][39][40] It has previously been demonstrated that alkoxyamines undergo charge-remote homolysis of the O-C bond. [41][42][43] For example, Oh et al derivatized small peptides with alkoxyamines [44][45][46] and subjected these to electrospray ionisation (ESI) to form ions with the charge sites localised to amino acid residues and thus remote from the alkoxyamine. Subsequent collisioninduced dissociation (CID) of these ions resulted in almost exclusive O-C bond homolysis, generating an alkyl radical which initiated further fragmentation of the peptide ion.…”