Nonergodic and conformational control of the electron capture dissociation of protein cations

Abstract

Electron capture dissociation (ECD) MS is proving to be unusually valuable for ''top down'' protein sequencing and identification͞ localization of posttranslational modifications, because the ECD product ions can represent cleavages between most of a protein's amino acids. As proposed, this unusual reactivity results from immediate local utilization, before randomization, of much of the relatively large (Ϸ6 eV) energy from the electron reaction with the multiply charged protein ion, minimizing the effect of differences in the backbone bond dissociation energies. However, others conclude that e ؊ capture produces a labile free radical species for which backbone cleavage is the lowest energy reaction. Supporting the nonergodic mechanism, ECD of ubiquitin (M ؉ 12H) 12؉ ions also yields thermalized radical (M ؉ 12H) 11؉⅐ ions that instead lose H· when activated. Also, the ECD spectrum of ubiquitin (M ؉ 13H) 13؉ ions is nearly unchanged by heating from 25°C to 125°C, demonstrating that this increase in thermal energy is small compared to the energy driving the reaction. These results support initial capture of the electron in a long-lived high-n Rydberg state, followed by internal conversion to the product valence state at an energy well above the dissociation barriers. The instantaneous conformation of the valence state is critical, with the observed products supporting an ␣-helical structure in which the protonated side chain of each basic residue is intercalated to hydrogen-bond to as many as three amide carbonyls. Activation (e.g., heat, collisions, lowered charge) can disrupt this conformation to allow additional H ؉ -side-chain interactions and provide more complete sequence coverage. E lectron capture dissociation (ECD) mass spectra provide far more extensive characterization of the primary structure of proteins, such as posttranslational modifications, than conventional methods for the dissociation of multiply charged protein cations (1-6). ECD products represent single cleavages between an unusually high proportion of the backbone amino acids (7, 8) (Fig. 1). To explain how hundreds of such reactions are apparently competitive, we have postulated (1-4) that ECD is a nonergodic process, a unimolecular dissociation that occurs before the excitation energy is randomized (9-13). Laser techniques can make such studies in vibrational time periods (11), although energy randomization can require 200 ps (12). Nonergodic dissociation has been observed only for small molecules with a relatively few internal degrees of freedom (9-11); the largest ion found to undergo nonergodic dissociation is the enolic acetone cation, CH 3 OC(OH)ACH 2 ϩ⅐ (12-15). Thus it is not surprising that substantial doubts have been expressed concerning our nonergodicity claim for protein ECD, instead presenting evidence that backbone cleavage of peptide ions from e Ϫ capture is just a free radical reaction of unusually low activation energy (16-21). Here we describe additional experiments that provide evidence for the high energy required and f...

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.…”

“…Supplementary Figure S2 demonstrates preferential cleavage at the NOC ␣ bond number 4 (z nϪ3 ) for fragments with a distinct ␣-helical or turn structure at the N-terminus (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] -118]). The periodicity is more pronounced in AI-ECD data ( Figure 5), demonstrating a period of 3 amino acids for the specified myoglobin fragments.…”

“…In ECD/ETD, interaction of a multiply charged peptide or protein ion with an electron/anion results in cleavage of NOC ␣ bonds along the peptide backbone, including chargesite-remote backbone cleavages [2]. ECD is useful for peptide sequence tag generation on a chromatographic timescale for protein identification in bottom-up mass spectrometry [6], de novo peptide sequencing [7], recognition of amino acid chirality [8], location and structural determination of single and multiple posttranslational modifications (PTMs) in peptides [9,10], sequencing and PTM analysis of large proteins (up to 50 -60 kDa) [11], and providing insight into protein secondary and tertiary structures in the gas phase [12,13]. ETD has already demonstrated high potential in several ECD application areas and continues to gain attention in peptide and protein structural analysis, especially in (phospho)proteomics [5,14,15].…”

“…Nevertheless, product ion abundance (PIA) distribution in ECD of peptides is believed to depend on peptide secondary structure in the gas phase [13]. Recent experimental and theoretical progress confirms the important role of hydrogen bonds in the backbone cleavage mechanism [21], allows better prediction of ECD product ion distribution [22][23][24], and establishes correlation with gas-phase peptide and protein structures [12,25,26]. However, the high structural complexity of peptides and proteins limits computational capabilities [21] and complicates analysis of the experimental results.…”

Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy

“…This method induces peptide fragmentation, primarily at N-C␣ bonds, using low-energy electrons [9]. Discussion continues as to the precise nature of the fragmentation mechanisms induced by ECD [10,11,12], but clearly the radical-induced fragmentations occur at low internal energy and are qualitatively different to those induced by CAD [13]. For instance, facile neutral loss of phosphoric acid, as commonly observed in CAD spectra of phosphopeptides, is rarely observed using ECD.…”