Electron capture dissociation product ion abundances at the X amino acid in RAAAA-X-AAAAK peptides correlate with amino acid polarity and radical stability

Abstract

We present mechanistic studies aimed at improving the understanding of the product ion formation rules in electron capture dissociation (ECD) of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry. In particular, we attempted to quantify the recently reported general correlation of ECD product ion abundance (PIA) with amino acid hydrophobicity. The results obtained on a series of model H-RAAAAXAAAAK-OH peptides confirm a direct correlation of ECD PIA with X amino acid hydrophobicity and polarity. The correlation factor (R) exceeds 0.9 for 12 amino acids (Ile, Val, His, Asn, Asp, Glu, Gln, Ser, Thr, Gly, Cys, and Ala). The deviation of ECD PIA for seven outliers (Pro is not taken into consideration) is explained by their specific radical stabilization properties (Phe, Trp, Tyr, Met, and Leu) and amino acid basicity (Lys, Arg). Phosphorylation of Ser, Thr, and Tyr decreases the efficiency of ECD around phosphorylated residues, as expected. R evealing peptide and protein structure-activity and structure-function relationships is an important and challenging step in the drug discovery process. Further insights into the influence of specific amino acids on molecular conformation and physicochemical properties are needed to increase the throughput and accuracy of the methods and techniques used to investigate these critical relationships [1,2]. Electron capture dissociation (ECD) [3] and electron-transfer dissociation (ETD) [4,5] are generally employed to determine peptide and protein primary structures and to characterize labile modifications in tandem mass spectrometry (MS/MS)-based proteomics. In addition, recent findings demonstrate that the probability of a given N-C ␣ bond in a peptide backbone being cleaved by ECD/ETD, which can be monitored by product ion abundance (PIA), is governed by peptide or protein conformation [6 -9]. A number of experiments ranging from targeted peptide analysis to statistical interpretation of fragmentation have been performed for several thousand tryptic peptides [6, 7, 10 -13] to examine the dependence of ECD PIA on peptide sequence. The experimental results have confirmed the complementarity of ECD and vibrationally induced dissociation, e.g., collision-induced dissociation (CID), but have failed to provide a general quantitative model for ECD. Enhancing models to include intramolecular hydrogen bonding has provided the best quantitative description of ECD PIA to date, as shown by Zubarev and coworkers [14]. However, their reported correlation is built upon extensive molecular dynamics simulations performed for a specific case of a selected part of a small (20-amino acid) protein, Trp cage. Although general application of the model would be time-consuming, it demonstrates the importance of hydrogen bonds when attempting to quantitatively describe ECD. Recent molecular dynamics modeling complemented with quantum chemistry calculations on penta-and hexapeptides supports the hypothesis that ECD/ETD possess strong conformational selectivity [15]. The ...

Effects of Peptide Backbone Amide-to-Ester Bond Substitution on the Cleavage Frequency in Electron Capture Dissociation and Collision-Activated Dissociation

“…Recently, the Tsybin group has demonstrated correlation between ECD cleavage frequency and stability of formed radical products [50]. Previously, product stability has often been suggested in theoretical literature as one of the major driving forces behind the cleavage of a particular bond (e.g., [48]).…”

Radical Stability Directs Electron Capture and Transfer Dissociation of β‐Amino Acids in Peptides

“…Although the radical stability of amino acids is known to direct ECD/ETD of a-amino acids and a-peptides, it is difficult to distinguish its influence from other effects (e.g., hydrogen bonding). [27] Interestingly, multiple additions of CH 2 groups into the peptide backbone shift the preferred product ion formation to the minor fragmentation channel observed in a-peptides.…”

“…The resulting ECD and ETD fragmentation patterns strongly differ from those reported for the a-analogues of these model peptides. [27] The extensive c-ion series, as well as the high mass-to-charge ratio z ions, are replaced by an almost complete radical a-ion series for all variants. High mass-to-charge ratio y ions are observed as well, and replace the corresponding z ions observed for a-peptides.…”

“…[11,12] Mentioned above as an exception, b 3 -Leu may also provide radical stabilization, as recently confirmed for radical-driven tandem mass spectrometry of peptides containing Leu by Julian and Tsybin. [27,33] Although Leu is similar to Ile, its side chain provides a stronger radical stabilization, due to the possible formation of a tertiary radical versus a secondary radical for isoleucine. [23] Nevertheless, the intrinsic radical stabilization of leucine (Scheme 4) does not seem to be sufficient, as no NÀC b bond cleavage has been observed for b 3 or b 2 -homo leucine substitutions in substance P. However, the case of the z 8 product ion retention in the LLLLALLLK À NH 2 peptide with b 3 -Leu 2 could indicate a more complex combination of radical stabilization and structural effects on ECD/ETD fragmentation inside b-amino acids.…”

The Early Life of a Peptide Cation-Radical. Ground and Excited-State Trajectories of Electron-Based Peptide Dissociations During the First 330 Femtoseconds

“…Peptide bond cleavages occur mainly, although not exclusively, between the amide nitrogens and adjacent C α atoms (N-C α cleavage for short) to form series of N-terminal (c) and C-terminal (z) fragment ions that are utilized for peptide and protein sequencing by mass spectrometry [1,4]. The nature of ExD has been investigated by experiment and theory with aims that ranged from statistical evaluation of the observed dissociations over large ad hoc sets of peptide ions [5,6] to analysis of fragmentations of selected model peptide ions to formulate plausible mechanisms [7][8][9][10][11][12][13][14][15][16][17][18].…”