Investigation of the mechanism of electron capture and electron transfer dissociation of peptides with a covalently attached free radical hydrogen atom scavenger

Abstract

The mechanisms of electron capture and electron transfer dissociation (ECD and ETD) are investigated by covalently attaching a free-radical hydrogen atom scavenger to a peptide. The 2,2,6,6-tetramethylpiperidin-l-oxyl (TEMPO) radical was chosen as the scavenger due to its high hydrogen atom affinity (ca. 280 kJ/mol) and low electron affinity (ca. 0.45 ev), and was derivatized to the model peptide, FQXTEMPOEEQQQTEDELQDK. The XTEMPO residue represents a cysteinyl residue derivatized with an acetamido-TEMPO group. The acetamide group without TEMPO was also examined as a control. The gas phase proton affinity (882 kJ/mol) of TEMPO is similar to backbone amide carbonyls (889 kJ/mol), minimizing perturbation to internal solvation and sites of protonation of the derivatized peptides. Collision induced dissociation (CID) of the TEMPO tagged peptide dication generated stable odd-electron b and y type ions without indication of any TEMPO radical induced fragmentation initiated by hydrogen abstraction. The type and abundance of fragment ions observed in the CID spectra of the TEMPO and acetamide tagged peptides are very similar. However, ECD of the TEMPO labeled peptide dication yielded no backbone cleavage. We propose that a labile hydrogen atom in the charge reduced radical ions is scavenged by the TEMPO radical moiety, resulting in inhibition of N−Cα backbone cleavage processes. Supplemental activation after electron attachment (ETcaD) and CID of the charge-reduced precursor ion generated by electron transfer of the TEMPO tagged peptide dication produced a series of b + H (bH) and y + H (yH) ions along with some c ions having suppressed intensities, consistent with stable O−H bond formation at the TEMPO group. In summary, the results indicate that ECD and ETD backbone cleavage processes are inhibited by scavenging of a labile hydrogen atom by the localized TEMPO radical moiety. This observation supports the conjecture that ECD and ETD processes involve long-lived intermediates formed by electron capture/transfer in which a labile hydrogen atom is present and plays a key role with low energy processes leading to c and z ion formation. Ab initio and density functional calculations are performed to support our conclusion, which depends most importantly on the proton affinity, electron affinity and hydrogen atom affinity of the TEMPO moiety.

The effect of micellization on the EPR spectra and reactivity of 2,2,4,4-tetramethylpiperidinoxyl (TEMPO) radicals

“…was the preferred one in the Stern layer . Their conclusion finds support in the observation that the gas phase proton affinity (PA) of TEMPO (882 kJ/mol) is similar to amide carbonyls (889 kJ/mol) . In addition, calculations on the basicity of TEMPO and related nitroxide radicals indicated that the PA of TEMPO derivatives like TEMPONE or TEMPOL, functionalized at the 4‐position, should be smaller than that of TEMPO.…”

Replacing H+by Na+or K+in phosphopeptide anions and cations prevents electron capture dissociation

“…To this end, we have studied both the electron capture efficiency and the efficiency of fragmentation into c , z ˙ or c ˙, z fragments by N–Cα backbone bond cleavage of peptide cations (2+, 3+) and anions (2–) with up to four Na + or K + ions attached. As the model peptide, we used bovine β-casein peptide F48–K63 (FQpSEEQQQTEDELQDK, 16 residues, referred to hereafter by their indices 1–16) that was previously shown to undergo dissociation into c , z ˙ or c ˙, z fragments by ECD, 29 – 31 ETD, 32 and niECD. 20 This peptide is phosphorylated (pS3) and has two basic (N terminus and K16) and eight acidic (pS3, E4, E5, E10, D11, E12, D15, and the C terminus) sites.…”

Free radical‐initiated peptide sequencing (FRIPS)‐based cross‐linkers for improved peptide and protein structure analysis

“…This finding suggests a non‐ergodic character of electron‐induced dissociation reactions, because these PTMs are weakly bound to the amino acid side chains and are therefore lost upon CID. In case of ECD and ETD however, the labile bonds survive activation, while backbone cleavage of the peptide occurs . Nevertheless, a number experimental and theoretical studies contradict this assumption suggesting that long‐lived intermediates are formed during ECD and ETD fragmentation processes and that IVR is also relevant for open‐shell peptide ion chemistry.…”

“…In case of ECD and ETD however, the labile bonds survive activation, while backbone cleavage of the peptide occurs. [45][46][47][48][49][50][51] Nevertheless, a number experimental and theoretical studies 35,40,44,[49][50][51][52] contradict this assumption suggesting that long-lived intermediates are formed during ECD and ETD fragmentation processes and that IVR is also relevant for open-shell peptide ion chemistry.…”