Effects of charge state and cationizing agent on the electron capture dissociation of a peptide

Abstract

Electron capture dissociation (ECD) is a promising method for de novo sequencing proteins and peptides and for locating the positions of labile posttranslational modifications and binding sites of noncovalently bound species. We report the ECD of a synthetic peptide containing 10 alanine residues and 6 lysine residues uniformly distributed across the sequence. ECD of the (M + 2H)(2+) produces a limited range of c (c(7)-c(15)) and z (z(9)-z(15)) fragment ions, but ECD of higher charge states produces a wider range of c (c(2)-c(15)) and z (z(2)-z(6), z(9)-z(15)) ions. Fragmentation efficiency increases with increasing precursor charge state, and efficiencies up to 88% are achieved. Heating the (M + 2H)(2+) to 150 degrees C does not increase the observed range of ECD fragment ions, indicating that the limited products are due to backbone cleavages occurring near charges and not due to effects of tertiary structure. ECD of the (M + 2Li)(2+) and (M + 2Cs)(2+) produces di- and monometalated analogues of the same c and z ions observed from the (M + 2H)(2+), with the abundance of dimetalated fragment ions increasing with fragment ion mass, a result consistent with the metal cations being located near the peptide termini to minimize Coulombic repulsion. In stark contrast to the ECD results, collisional activation of cesiated dications overwhelmingly results in ejection of Cs(+). The abundance of cesiated fragment ions formed from ECD of the (M + Cs + Li)(2+) exceeds that of lithiated fragment ions by 10:1. ECD of the (M + H + Li)(2+) results in exclusively lithiated c and z ions, indicating an overwhelming preference for neutralization and cleavage at protonated sites over metalated sites. These results are consistent with preferential neutralization of the cation with the highest recombination energy.

Figure 1

ESI mass spectra of 6KI (10⁻⁵ M) formed from 47% water/ 50% methanol/3% acetic acid with (a) 0 and (b) 0.5% m-nitrobenzyl alcohol. The capillary exit voltage, second skimmer voltage, and hexapole offset for (a) and (b) are 150 and 100, 8.0 and 9.0, and 5.0 and 4.0 V, respectively. Peaks due to impurities in the sample, e.g., 6KI lacking one or two lysine residues, and peaks arising from sodium adduction are present in the mass spectra (the sample was not purified after synthesis).

Figure 2

ECD spectra of the (a) (M + 2H)²⁺, (b) (M + 3H)³⁺, (c) (M+ 4H)⁴⁺, and (d) (M + 5H)⁵⁺ of 6KI. The “L” denotes C₆H₁₃N₂O⁺ (m/z 129), presumably a product of internal cleavage and rearrangement of a lysine residue. Noise peaks are indicated by asterisks.

Figure 3

ECD fragment ion plots of the (a) (M + 2H)²⁺, (b) (M + 3H)³⁺, (c) (M + 4H)⁴⁺, and (d) (M + 5H)⁵⁺ of 6KI.

Figure 4

Spectra of the (M + 2H)²⁺ collected at (a) 100 °C with ECD, (b) 150 °C with ECD, and (c) 150 °C without ECD. The peaks at m/z 129 and 84 are C₆H₁₃N₂O⁺ and C4H6NO⁺, respectively. Noise peaks are indicated by asterisks (the heating circuit used for the heated cell experiments results in spurious noise peaks).

Figure 5

Mechanism for ECD of the (M + 2H)²⁺. The charges (protonated lysine side chains) are located near the termini of the peptide and are solvated by backbone carbonyl oxygens. Electron capture and neutralization of the charge near the C-terminus (a) results in formation of a high-mass c ion and a low-mass, neutral z product. Similarly, neutralization of the charge near the N-terminus (b) produces a high-mass z ion and a low-mass, neutral c product. (The neutral products are not shown.)

Figure 6

ECD spectra of the (a) (M + 2Li)²⁺ and (b) (M + 2Cs)²⁺.

Figure 7

ECD fragment ion plots of the (a) (M + 2Li)²⁺ and (b) (M + 2Cs)²⁺.

Figure 8

ECD spectra of the (a) (M + Cs + Li)²⁺ and (b) (M + H + Li)²⁺.

Figure 9

ECD fragment ion plots of the (a) (M + Cs + Li)²⁺ and (b) (M + H + Li)²⁺.

Figure 10

SORI-CAD spectra of the (a) (M + 2Li)²⁺, (b) (M + 2Cs)²⁺, and (c) (M + Cs + Li)²⁺. The peak at m/z 135 in (a) is LiC₆H₁₂N₂O⁺.