Conformation of Polyalanine and Polyglycine Dications in the Gas Phase: Insight from Ion Mobility Spectrometry and Replica-Exchange Molecular Dynamics

The conformation of model Arg(Ala)(4)X(Ala)(4)Lys+2H and Arg(Gly)(4)X(Gly)(4)Lys+2H peptides has been systematically investigated as a function of the central amino acid X through a combined experimental and theoretical approach. Mass spectrometry-based ion mobility measurements have been performed together with conformational sampling using replica-exchange molecular dynamics to probe the influence of each amino acid on the stable peptide conformation. Satisfactory agreement is obtained between measured and calculated diffusion cross section distributions. The results confirm the propensity of alanine-based peptides to form a-helices in the gas phase, differences between peptides arising from the local arrangement of the central side chain with respect to the charged ends. More generally, we find that charge solvation plays a major role in secondary structure stabilization, especially in the case of glycine-based peptides. The rich variety of conformations exhibited by the latter is qualitatively captured by the simulations. This work illustrates the potentiality of such combined experimental/theoretical strategy to determine peptide secondary structures. The present polyalanine and polyglycine peptides also offer a series of benchmark systems for future conformation-resolved studies.

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

Structural Preferences of Gas-Phase M2TMP Monomers upon Sequence Variations

Rodolphe Antoine, Hisham Ben Hamidane, Yury Tsybin

The conformations of a number of M2TMP-(22-46) sequence variants have been investigated using ion mobility spectrometry (IMS). Substantial conformational changes were evidenced by IMS upon the variation of a single amino acid in the peptide sequence, with two main drift time signatures. Replica-exchange molecular dynamics simulations were used to help assign the structures of the different identified conformers. Even though one-on-one agreement with experiment was found for only two variants, the simulations generally confirmed the existence of two structural families. Based on these results, most of the triply protonated variants, including the wild-type peptide, were found to display collision cross sections in agreement with compact conformations in the gas phase, whereas they tend to form extended alpha-helices in the condensed phase, as confirmed by circular dichroism and previously reported NMR measurements. The destabilization of alpha-helices in vacuo upon amino acid substitution is interpreted as being driven by the solvation pattern of the charges.

2011

A Combination of Cold Ion Spectroscopy and Ion Mobility for the Study of Complex Peptides and Small Proteins

Georgios Papadopoulos

We present in this work experiments that push the limits of cold ion spectroscopy to the study of complex peptides and small proteins in the gas phase. Although the low temperature attainable in a cold ion trap greatly simplifies the electronic spectra of large molecules, conformational heterogeneity can still be a significant source of congestion, complicating spectroscopic analysis. To overcome this hurdle, we propose an in tandem combination of ion mobility spectrometry and photofragment spectroscopy. This permits for conformational separation prior to laser interrogation. Towards this end, we describe a proof-of-principle experiment where we combine high- Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) with electronic spectroscopy. We demonstrate that using FAIMS to separate gas-phase peptide conformers before injecting them into a cold ion trap allows one to decompose a dense spectrum into contributions from different conformational families. In the inverse sense, cold ion spectroscopy can be used as a conformation-specific detector for ion mobility, allowing one to separate an unresolved mobility peak into contributions from different conformational families. The doubly protonated peptide bradykinin serves as a good test case for the marriage of these two techniques as it exhibits a considerable degree of conformational heterogeneity that results in a highly congested electronic spectrum. Our results demonstrate the feasibility and advantages of directly coupling ion mobility with spectroscopy and provide a diagnostic of conformational isomerization of this peptide after being produced in the gas phase by electrospray. In a second series of studies we show the potential but also the limitations of our spectroscopic approach for investigating small, naturally occurring proteins, by applying it to ubiquitin, a protein of 76 amino acids. We present electronic photofragment spectra of various protonation states of ubiquitin and we compare our results to literature data acquired by ion mobility spectrometry. These experiments show that the information obtained by laser interrogation of molecules of this size is limited and could be enhanced by a combination with ion mobility techniques. Although some of the results obtained may be important in characterizing small proteins in the gas phase, these experiments are at an early stage and the application of photofragment spectroscopy on proteins needs more investigation.