The effect of fixed charge modifications on electron capture dissociation

Abstract

Electron capture dissociation (ECD) studies of two modified amyloid beta peptides (20-29 and 25-35) were performed to investigate the role of H* radicals in the ECD of peptide ions and the free-radical cascade (FRC) mechanism. 2,4,6-Trimethylpyridinium (TMP) was used as the fixed charge tag, which is postulated to both trap the originally formed radical upon electron capture and inhibit the H* generation. It was found that both the number and locations of the fixed charge groups influenced the backbone and side-chain cleavages of these peptides in ECD. In general, the frequency and extent of backbone cleavages decreased and those of side-chain cleavages increased with the addition of fixed charge tags. A singly labeled peptide with the tag group farther away from the protonated site experienced a smaller abundance decrease in backbone cleavage fragments than the one with the tag group closer to the protonated site. Despite the nonprotonated nature of all charge carriers in doubly labeled peptide ions, several c and z* ions were still observed in their ECD spectra. Thus, although H* transfer may be important for the NC(alpha) bond cleavage, there also exist other pathways, which would require a radical migration via H* abstraction through space or via an amide superbase mechanism. Finally, internal fragment ions were observed in the ECD of these linear peptides, indicating that the important role of the FRC in backbone cleavages is not limited to the ECD of cyclic peptides.

Figure 1

ECD spectra of the (a) unlabeled, (b) singly-labeled and (c) doubly-labeled Amyloid β peptide (20-29). * marks the electronic noise peak, ω2 marks the first harmonic peak of the precursor ion, peaks marked with “–amino acid residue” resulted from cleavage at the C_α−C_b bond, and partial side-chain losses were represented by molecular formula of the departing group(s). Tagged precursor ions were labeled as [Mt¹+H]²⁺ and [Mt²]²⁺ for singly- and doubly-labeled peptides, respectively. Cleavage patterns are shown as the insets.

Figure 2

ECD spectra of the (a) unlabeled, (b) singly-labeled and (c) doubly-labeled Amyloid β peptide (25–35). Peak labeling follows the same convention as in Figure 1. Cleavage patterns are shown as the insets.

Figure 3

All observed ECD cleavages of the (a) unlabeled, (b) singly-labeled and (c) doubly-labeled Ab peptide (20–29).

Figure 4

All observed ECD cleavages of the (a) unlabeled, (b) singly-labeled and (c) doubly-labeled Ab peptide (25–35).

Scheme 1

The ε-amino group of the lysine side-chain was selectively converted to a 2,4,6-trimethylpyridinium salt.

Scheme 2

H loss from the TMP tag upon electron capture.

Scheme 3

One possible mechanism for c/z ion formations in doubly tagged peptides.

Scheme 4

Tag loss after the electron capture.

Scheme 5

Methyl group loss from the tag loss product.

Scheme 6

Methyl loss from the valine (R = CH₃) and isoleucine (R = CH₂CH₃) side-chains.

Scheme 7

Methionine side-chain losses from z₈ ion.