Heterolytic N-Calpha Bond Cleavage in Electron Capture and Transfer Dissociation of Peptide Cations

Mass spectrometry techniques employing electron capture and electron transfer dissociation represent powerful approaches for the analysis of biological samples. Despite routine employment in analytical fields, the underlying physical processes dictating peptide fragmentation remain less understood. Among the most accepted mechanisms, the Cornell proposal of McLafferty postulates that the homolytic cleavage of N-C-alpha bonds located in the peptide backbone occurs on the right (C-terminal) side of a hydrogen acceptor carbonyl group. Here, we illustrate that an alternative "enol" mechanism, based on a hetetolytic N-C-alpha bond cleavage located on the left (N-terminal) side of an acceptor carbonyl group, not only is thermodynamically viable but also often represents the energetically preferred cleavage route.

On the Viability of Heterolytic Peptide N-Calpha Bond Cleavage in Electron Capture and Transfer Dissociation Mass Spectrometry

Yury Tsybin, Matthew Wodrich, Konstantin Zhurov

While frequently employed as an experimental technique, the mechanistic picture surrounding the gas-phase dissociation of peptides carrying multiple positive charges during electron capture and electron transfer dissociation tandem mass spectrometry remains incomplete. Despite this mechanistic uncertainty, most proposals agree that the peptide backbone N-C-alpha bond located to the C-terminal (right) side of an aminoketyl radical formed in a peptide backbone during the electron capture process is homolytically cleaved. Recently, we introduced the "enol" mechanism, which proposes that a backbone N-C-alpha bond located to the N-terminal (left) side of an aminoketyl radical is cleaved heterolytically. Here, we further validate this mechanism using replica-exchange molecular dynamics to create unbiased representative sets of low-energy conformers for several model tryptic peptide systems (H-Ala(x)-Lys-OH2+, x = 3-5). Transition state barrier enthalpies for the cleavage of N-C-alpha bonds proceeding via the homolytic (right-side) and heterolytic (left-side) pathways, determined by density functional computations, identify the preferred cleavage route for each conformer. These findings support our original hypothesis that heterolytic N-C-alpha cleavage can exist in a competitive balance with homolytic cleavages, independent of the relative energy of the precursor dication species. Smaller peptide systems see decreased heterolytic N-C-alpha cleavage probabilities, likely resulting from an insufficient hydrogen-bonding network needed to stabilize and ultimately annihilate the transition state zwitterion. This observation may explain the early dismissal of left-side cleavage pathways based on computational studies employing small model systems.

Amer Chemical Soc2014

Peptide Structure and Radical Chemistry in the Gas Phase Revealed by Product Ion Abundance Analysis in Electron Capture and Transfer Dissociation Mass Spectrometry

Hisham Ben Hamidane

Peptide and protein ion abundances in mass spectrometry (MS) and tandem mass spectrometry (MS/MS) may provide orthogonal, or complementary, information to their mass measurements. Collision induced dissociation (CID), the most commonly used MS/MS technique, demonstrates high variability of fragmentation pathways and their relative product abundances as a function of experimental parameters. Moreover, a reliable prediction of relative abundances of CID products is possible only for relatively small systems, but not for larger biomolecules. Recently developed radical-induced fragmentation techniques, e.g., electron capture and transfer dissociation (ECD/ETD), appear to demonstrate more repeatable and reproducible fragmentation patterns. However, it has been reported that ECD/ETD product ion abundance distribution for peptides is rather flat and extracting additional peptide sequence information is yet to be done. In the present work, an attempt is made to understand the peptide's ECD/ETD product ion abundance formation fundamentals and to reveal the possible implications of this source of information for improving peptide and protein structural characterization. Specifically, we have shown that ECD/ETD of peptides is sensitive to the chemical environment in terms of peptide primary structure (amino acid nature) and possibly higher order structure (hydrogen bond connectivity). After demonstrating the analytical validity of the ECD/ETD product ion abundance methodology, its parameter dependence and its applicability to different experimental setups, results will be presented and rationalized along two main axes of comprehension: gas phase peptide radical chemistry and structure. Whereas fundamental aspects of ECD/ETD (mechanism, thermodynamics) can be addressed with the former, additional ECD/ETD yield modulations were observed and presumably relate to structural properties of ions in the gas phase. ECD specificity will be demonstrated on the class of amphipathic peptides, where periodicity is revealed in product ion abundance and correlated to peptide primary and secondary structure. To decipher between both contributions, sequence variations of amphipathic peptides were probed by ECD and correlated to gas phase structures suggested by ion mobility mass spectrometry and supported by molecular dynamics computations. After probing amino acid side chain influence, unraveling the chemical environment specificity of ECD/ETD led us to investigate peptide backbone modifications. Peptides containing beta amino acids revealed two main effects in ECD and ETD. We demonstrate that individual substitutions yield c and z type fragments only for amino acids that provide sufficient z• radical stabilization, whereas multiple substitutions shift the preferential fragmentation pathway to the otherwise minor a-type ion formation pathway indicating possible change in peptide protonation. Finally, understanding the partitioning of radical ions between N- and C-terminal fragments led us to investigate ion internal energy dependence. The extent of hydrogen atom transfer, the [c+z] complex lifetime and secondary fragmentation pathways (w-type ion formation) are directly modulated by ion internal energy as demonstrated by decoupling ETD from charge reduced CID. To summarize, ECD/ETD product ion abundance methodology developed in the thesis advances biomolecule radical chemistry understanding and provides a potentially useful methodology for peptide and protein structural characterization.

EPFL2011

Ping-Pong Protons: How Hydrogen Bonding Networks Facilitate Heterolytic Bond Cleavages in Peptide Radical Cations

Yury Tsybin, Matthew Wodrich, Konstantin Zhurov

Electron capture and electron transfer dissociation (ECD/ETD) tandem mass spectrometry (MS/MS) are commonly employed techniques for biomolecular analysis. The ECD/ETD process predominately cleaves N-C-alpha peptide backbone bonds, leading to primary sequence information complementary to other mass spectrometry techniques. Despite frequent laboratory use, the mechanistic underpinnings surrounding N-C-alpha, bond cleavage remain debated. While the majority of mechanisms assume a homolytic bond rupture, we recently showed that heterolytic cleavage is also thermodynamically viable. For a cleavage of this type to be feasible, the charge separation created upon breaking of the N-C-alpha backbone bond must be quickly annihilated. In this work, we show, using density functional computations, that specific hydrogen-bonding motifs and structural rearrangements involving proton transfers stabilize the transition state associated with heterolytic cleavage and eliminate the ensuing charge separation from the final product fragments. The movement of protons can occur either directly from the z- to c-fragment or in a more complex manner including a ping-pong-type mechanism. The nature of these diverse hydrogen-bonding motifs reveals that not only those functional groups proximate to the bond rupture site, but also the entire global chemical environment, play important roles in backbone cleavage characteristic of ECD/ETD MS/MS. For doubly charged systems, both conformation and electron localization site dictate which of the two fragments retains the final positive charge.

Amer Chemical Soc2014

Peptide Structure and Radical Chemistry in the Gas Phase Revealed by Product Ion Abundance Analysis in Electron Capture and Transfer Dissociation Mass Spectrometry

Hisham Ben Hamidane

EPFL2011