Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors

Abstract

Electron-transfer dissociation (ETD) delivers the unique attributes of electron capture dissociation to mass spectrometers that utilize radio frequency trapping-type devices (e.g., quadrupole ion traps). The method has generated significant interest because of its compatibility with chromatography and its ability to: (1) preserve traditionally labile post-translational modifications (PTMs) and (2) randomly cleave the backbone bonds of highly charged peptide and protein precursor ions. ETD, however, has shown limited applicability to doubly protonated peptide precursors, [M + 2H]2+, the charge and type of peptide most frequently encountered in "bottom-up" proteomics. Here we describe a supplemental collisional activation (CAD) method that targets the nondissociated (intact) electron-transfer (ET) product species ([M + 2H]+*) to improve ETD efficiency for doubly protonated peptides (ETcaD). A systematic study of supplementary activation conditions revealed that low-energy CAD of the ET product population leads to the near-exclusive generation of c- and z-type fragment ions with relatively high efficiency (77 +/- 8%). Compared to those formed directly via ETD, the fragment ions were found to comprise increased relative amounts of the odd-electron c-type ions (c+*) and the even-electron z-type ions (z+). A large-scale analysis of 755 doubly charged tryptic peptides was conducted to compare the method (ETcaD) to ion trap CAD and ETD. ETcaD produced a median sequence coverage of 89%-a significant improvement over ETD (63%) and ion trap CAD (77%).

Figure 1

Product ion spectra following ETD of the triply (A) and doubly (B) protonated synthetic peptide KAAAKAAAK. Note the dication generates only half of the possible c and z-type product ions.

Figure 2

Isotopic distribution of a charge reduced, non-dissociated ET product (A, [M+2H]^+•) and the corresponding singly protonated version (B, [M+H]⁺). The fragility of the ET product results in low resolution during mass analysis as compared to its even electron counterpart.

Figure 3

Contour maps plotting the effect of activation q_u-value and excitation voltage for collisional activation of an ET product ion ([M+2H]^+•). Panel A displays the abundance of the intact ET product (the ion subjected to secondary activation); panel B plots the formation of an ETD-type product ion (c₁₀) not produced directly by ETD; panel C displays formation of a CAD-type product ion (b₈). Plotted to the right are three two-dimensional cross-sections (varying excitation voltage at fixed q_u values of 0.15, 0.18, and 0.20) of this data. These cross-sections also plot the combined total product ion signal derived from ET, ETD and ETcaD. Note the total ion signal trace does not include the precursor signal. Because the ET precursor ion was not isolated, this signal begins at ~ 80% relative intensity – signal that can be attributed to c and z-type fragments generated directly by ETD.

Figure 4

Plot of the onset values (q_u and normalized collision energy) for observation of either CAD-type or ETD-type product ions following supplemental activation of six different intact ET product ion species.

Figure 5

Comparison of ETD (A–C) and ETcaD (D–F) tandem mass spectra (single scan) for 3 tryptic peptides. Each of these was acquired during a data-dependent analysis of a complex tryptic peptide mixture derived from Arabidopsis.

Figure 6

Fragmentation data array that summarizes the performance of CAD, ETD, and ETcaD for doubly charged peptide precursors ranging from 7 to 16 amino acids in length. Observed fragment ions from each peptide are depicted in two rows; N-terminal product ions are shown from left to right in blue, while C-terminal products are displayed from right to left in orange. Color intensity correlates with observed product ion intensity. Note for ETD a significant gap in sequence coverage becomes increasingly apparent as the peptide size increases. This gap is substantially minimized by use of ETcaD.

Figure 7

Percent sequence coverage resulting from CAD, ETD, and ETcaD as a function of precursor m/z (A) and frequency (B) for the 755 doubly protonated tryptic peptides studied here.

Scheme 1