Electron capture dissociation at low temperatures reveals selective dissociations

Abstract

Electron capture dissociation at 86 K of the linear peptide Substance P produced just two backbone fragments, whereas at room temperature eight backbone fragments were formed. Similarly, with the cyclic peptide gramicidin S, just one backbone fragment was formed at 86 K but five at room temperature. The observation that some backbone scissions are active and others inactive, when all involve N™C ␣ cleavages and have a high rate constant, indicates that the more specific fragments at low temperatures reflects the reduced conformation heterogeneity at low temperatures. This is supported by reduced or inactive hydrogen loss, a channel that has previously been shown to be affected by conformation. The conclusion that the ECD fragments are a snapshot of the conformational (intramolecular solvation shell) heterogeneity helps explain how the relative intensities of ECD fragments can be different on different instrument and highlights the common theme in methodologies used to increase sequence coverage, namely an increase in the conformational heterogeneity of the precursor ion population. ( ploys reactions of multiprotonated peptides/ proteins with low energy electrons to generate peptide fragments. The high sequence coverage and the ability to retain labile groups after ECD have allowed posttranslational modifications (PTMs) and point mutations (PMs) in peptides and proteins to be both identified and localized. Such performance is of great value for the analysis of the proteome, particularly that of diseased organisms. PTMs and PMs are widespread and are frequently associated with disease [5][6][7], for example over 80 different point mutations have been found in transthyretin (most of which lead to autosomal disorders) [8].To be able to fully characterize such modified proteins, 100% sequence coverage is required (so-called top-down proteomics). Several methods have been developed to increase the sequence coverage of ECD, which can be grouped into either infrared illumination [9 -11] or collisional activation [12]. These methods increase the average internal energy of the ions, which also affects the conformation of these gas-phase ions [13].There is experimental and theoretical evidence that ECD is directed by the internal solvation of the (neutralized) proton [9, 14 -18]. If the reaction progresses faster than the electron-proton recombination energy is randomized and thermal fluctuations are small, specific fragments would be expected from a single (frozen) conformer, reflecting the solvation shell, specific for that conformation, surrounding the neutralized proton. Thermal fluctuations that result in a dynamic solvation shell, but not conformational change (local fluctuations rather than a global change), would produce a greater number of fragments. Finally, multiple conformations as well as thermal fluctuations would produce yet more fragments. Because the methods used to increase the sequence coverage of ECD all increase the internal energy of the ions, and so affect the conformation of the gas-phase ions, the...

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.…”

“…Presumably, the Coulombic potential induced by protonation at His 16 and Arg 24 is responsible for this shift. Preferential fragmentation at the N-terminal side of Arg 24 and Asp 3 takes place outside of the ␣-helical part of M2 TMP and indicates preferential product ion formation near hydrophilic residues (Asp and Arg are among the most hydrophilic residues according to Hessa's scale and others).…”

“…Periodic distribution of PIA in ECD would then be explained by the characteristic location of not only basic, presumably protonated, amino acids (e.g., His 16 and Arg 24 ), but also neutral amino acids (e.g., glycine) that can allow exothermic abstraction of an ␣-proton toward the long-lived amide anion in an excited electronic state [56,57]. In M2 TMP, a Gly residue is located in the middle of the peptide, at position 12, one ␣-helix turn away from the basic His 16 residue and therefore fulfilling the periodic requirement.…”

“…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.…”