Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

Abstract

We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and [7]. In addition to mainly product ion mass-based MS/ MS, product ion abundance (PIA) in ECD is increasingly considered as a new source of information to improve peptide and protein sequencing [8,9], quantitative modification analysis [10, 11], higher-order structure characterization [8,[12][13][14][15], providing new insights into ECD mechanism [16,17], suggesting charge location in peptides and proteins [18,19], and indicating routes toward developing a quantitative model of ECD/ETD [15]. Double-resonance (DR) ECD, with and without ion preactivation, is used to estimate the radical intermediate lifetimes and differentiate between short-lived and long-lived intermediates by monitoring PIA variation attributed to radical intermediates ejection from the ICR trap immediately upon formation [20,21]. An alternative approach to DR-ECD is to compare ECD fragmentation patterns obtained at room-temperature (300 K) and cold (86 K) ICR ion trap conditions [22]. In cold ICR trap long-lived radical intermediates remain inside of the trap but do not have sufficient internal energy to initiate product ion separation and thus do not contribute to the product ion mass spectrum [9,22]. In general, long-lived radical intermediates exhibit a higher yield of radical N-terminal product ions, c· ions, and even-electron or prime C-terminal product ions, z= ions than that of short-lived intermediates, presumably as the result of increased probability of hydrogen atom transfer between ECD products [9,23]. Ion internal energy variation in activated ion (AI)-ECD [24] was shown to influence hydrogen atom rearrangement between ECD products and determine the ratio of radical to prime product ions [9,21]. Consideration of hydrogen atom loss/gain is important for correct product ion assignment and error-free peptide sequencing in proteomics [23,25].Implementation of electron-transfer dissociation (ETD) in io...

Influence of charge state and amino acid composition on hydrogen transfer in electron capture dissociation of peptides

“…The H t values of each peptide are shown in column 2 of [2,16,18]. Furthermore, it has been reported that collisional activation of the charge reduced species [M ϩ 2H] ϩ· can improve fragmentation efficiency and increase relative amounts of the c ϩ· and z= ϩ ions in ETD [19].…”

Periodic sequence distribution of product ion abundances in electron capture dissociation of amphipathic peptides and proteins

“…ECD FT-ICR MS of six doubly protonated horse myoglobin tryptic fragments ( [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16], [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], [32][33][34][35][36][37][38][39][40][41][42] Figure S2) and with vibrational activation ( Figure 5) confirm preferential and sometimes periodic product ion formation from peptides with mainly ␣-helical or ␤-turn secondary structure in solution (before protein digestion). Predominantly z-ions are observed because of preferential charge retention at the C-terminal Lys or Arg basic residue upon electron capture, as expected for doubly charged tryptic peptides.…”

“…Therefore, we selected the class of amphipathic peptides and proteins to probe ECD characteristics, to improve both fundamental and practical aspects of ECD in MS/MS. Projection of ECD Fourier transform ion cyclotron resonance (FT-ICR) MS results to ETD in ion trap MS is considered as a second step and will require estimation of ion cooling at the higher pressure of ETD experiments [36].…”

Mapping N-terminus phosphorylation sites and quantitation by stable isotope dimethyl labeling

“…Among these excluded sites, the site on Ser230 of G3BP1 and the site on Thr399 of SRC8 proteins were assigned to be phosphorylation sites in previous reports [29,30] without confirmation. It is worth noting that the synthetic peptides shown in Figures 2b and 3b without and with dimethyl labeling, respectively, were the same sequence (SSpSPAPADIAQTVQEDLR) derived from Ras-GTPase-activating protein-binding protein 1 as shown in Figure 4a, whereas, unlike those detected from the synthetic peptides, no y 16 or y 17 ions (Figure 4a) could be detected from A431 cells to help in identifying the exact phosphorylation site. Likewise, the synthetic peptide shown in Figure 3c is the same sequence (TQpTPPVSPAPQPTEER) derived from Src substrate cortactin as that displayed in Figure 4b but with different phosphorylation sites.…”