Nucleation Inhibition of Huntingtin Protein (htt) by Polyproline PPII Helices: A Potential Interaction with the N-Terminal α-Helical Region of Htt.

Huntington's disease is a genetic neurodegenerative disorder characterized by the formation of amyloid fibrils of the huntingtin protein (htt). The 17-residue N-terminal region of htt (Nt17) has been implicated in the formation of early phase oligomeric species, which may be neurotoxic. Because tertiary interactions with a downstream (C-terminal) polyproline (polyP) region of htt may disrupt the formation of oligomers, which are precursors to fibrillar species, the effect of co-incubation of a region of htt with a 10-residue polyP peptide on oligomerization and fibrillization has been examined by atomic force microscopy. From multiple, time-course experiments, morphological changes in oligomeric species are observed for the protein/peptide mixture and compared with the protein alone. Additionally, an overall decrease in fibril formation is observed for the heterogeneous mixture. To consider potential sites of interaction between the Nt17 region and polyP, mixtures containing Nt17 and polyP peptides have been examined by ion mobility spectrometry and gas-phase hydrogen-deuterium exchange coupled with mass spectrometry. These data combined with molecular dynamics simulations suggest that the C-terminal region of Nt17 may be a primary point of contact. One interpretation of the results is that polyP may possibly regulate Nt17 by inducing a random coil region in the C-terminal portion of Nt17, thus decreasing the propensity to form the reactive amphipathic α-helix. A separate interpretation is that the residues important for helix-helix interactions are blocked by polyP association.

Comparison of Peptide Ion Conformers Arising from Non-Helical and Helical Peptides Using Ion Mobility Spectrometry and Gas-Phase Hydrogen/Deuterium Exchange.

The dominant gas-phase conformer of [M+3H]3+ ions of the model peptide acetyl-PSSSSKSSSSKSSSSKSSSSK has been examined with ion mobility spectrometry (IMS), gas-phase hydrogen deuterium exchange (HDX), and mass spectrometry (MS) techniques. The [M+3H]3+ peptide ions are observed predominantly as a relatively compact conformer type. Upon subjecting these ions to electron transfer dissociation (ETD), the level of protection for each amino acid residue in the peptide sequence is assessed. The overall per-residue deuterium uptake is observed to be relatively more efficient for the neutral residues than for the model peptide acetyl-PAAAAKAAAAKAAAAKAAAAK. In comparison, the N-terminal and C-terminal regions of the serine peptide show greater relative protection compared with interior residues. Molecular dynamics (MD) simulations have been used to generate candidate structures for collision cross section and HDX reactivity matching. Hydrogen accessibility scoring (HAS) for select structural candidates from MD simulations has been used to suggest conformer types that could contribute to the observed HDX patterns. The results are discussed with respect to recent studies employing extensive MD simulations of gas-phase structure establishment of a peptide system. Graphical Abstract ᅟ.

Comprehensive Peptide Ion Structure Studies Using Ion Mobility Techniques: Part 3. Relating Solution-Phase to Gas-Phase Structures.

Molecular dynamics (MD) simulations have been utilized to study peptide ion conformer establishment during the electrospray process. An explicit water model is used for nanodroplets containing a model peptide and hydronium ions. Simulations are conducted at 300 K for two different peptide ion charge configurations and for droplets containing varying numbers of hydronium ions. For all conditions, modeling has been performed until production of the gas-phase ions and the resultant conformers have been compared to proposed gas-phase structures. The latter species were obtained from previous studies in which in silico candidate structures were filtered according to ion mobility and hydrogen-deuterium exchange (HDX) reactivity matches. Results from the present study present three key findings namely (1) the evidence from ion production modeling supports previous structure refinement studies based on mobility and HDX reactivity matching, (2) the modeling of the electrospray process is significantly improved by utilizing initial droplets existing below but close to the calculated Rayleigh limit, and (3) peptide ions in the nanodroplets sample significantly different conformers than those in the bulk solution due to altered physicochemical properties of the solvent. Graphical Abstract ᅟ.

Comprehensive Gas-Phase Peptide Ion Structure Studies Using Ion Mobility Techniques: Part 2. Gas-Phase Hydrogen/Deuterium Exchange for Ion Population Estimation.

Gas-phase hydrogen/deuterium exchange (HDX) using D2O reagent and collision cross-section (CCS) measurements are utilized to monitor the ion conformers of the model peptide acetyl-PAAAAKAAAAKAAAAKAAAAK. The measurements are carried out on a home-built ion mobility instrument coupled to a linear ion trap mass spectrometer containing electron transfer dissociation (ETD) capabilities. ETD is utilized to obtain per-residue deuterium uptake data for select ion conformers, and a new algorithm is presented for interpreting the HDX data. Using molecular dynamics (MD) production data and a hydrogen accessibility scoring (HAS)-number of effective collisions (NEC) model, hypothetical HDX behavior is attributed to various in-silico candidate (CCS match) structures. The HAS-NEC model is applied to all candidate structures, and non-negative linear regression is employed to determine structure contributions resulting in the best match to deuterium uptake. The accuracy of the HAS-NEC model is tested with the comparison of predicted and experimental isotopic envelopes for several of the observed c-ions. It is proposed that gas-phase HDX can be utilized effectively as a second criterion (after CCS matching) for filtering suitable MD candidate structures. In this study, the second step of structure elucidation, 13 nominal structures were selected (from a pool of 300 candidate structures) and each with a population contribution proposed for these ions. Graphical Abstract ᅟ.

Comprehensive Peptide Ion Structure Studies Using Ion Mobility Techniques: Part 1. An Advanced Protocol for Molecular Dynamics Simulations and Collision Cross-Section Calculation.

Collision cross-section (CCS) measurements with a linear drift tube have been utilized to study the gas-phase conformers of a model peptide (acetyl-PAAAAKAAAAKAAAAKAAAAK). Extensive molecular dynamics (MD) simulations have been conducted to derive an advanced protocol for the generation of a comprehensive pool of in-silico structures; both higher energy and more thermodynamically stable structures are included to provide an unbiased sampling of conformational space. MD simulations at 300 K are applied to the in-silico structures to more accurately describe the gas-phase transport properties of the ion conformers including their dynamics. Different methods used previously for trajectory method (TM) CCS calculation employing the Mobcal software [1] are evaluated. A new method for accurate CCS calculation is proposed based on clustering and data mining techniques. CCS values are calculated for all in-silico structures, and those with matching CCS values are chosen as candidate structures. With this approach, more than 300 candidate structures with significant structural variation are produced; although no final gas-phase structure is proposed here, in a second installment of this work, gas-phase hydrogen deuterium exchange data will be utilized as a second criterion to select among these structures as well as to propose relative populations for these ion conformers. Here the need to increase conformer diversity and accurate CCS calculation is demonstrated and the advanced methods are discussed. Graphical Abstract ᅟ.

Ion Mobility Spectrometry-Hydrogen Deuterium Exchange Mass Spectrometry of Anions: Part 2. Assessing Charge Site Location and Isotope Scrambling.

Ion mobility spectrometry (IMS) coupled with gas-phase hydrogen deuterium exchange (HDX)-mass spectrometry (MS) and molecular dynamic simulations (MDS) has been used for structural investigation of anions produced by electrospraying a sample containing a synthetic peptide having the sequence KKDDDDDIIKIIK. In these experiments the potential of the analytical method for locating charge sites on ions as well as for utilizing collision-induced dissociation (CID) to reveal the degree of deuterium uptake within specific amino acid residues has been assessed. For diffuse (i.e., more elongated) [M - 2H](2-) ions, decreased deuterium content along with MDS data suggest that the D4 and D6 residues are charge sites, whereas for the more diffuse [M - 3H](3-) ions, the data suggest that the D4, D7, and the C-terminus are deprotonated. Fragmentation of mobility-selected, diffuse [M - 2H](2-) ions to determine deuterium uptake at individual amino acid residues reveals a degree of deuterium retention at incorporation sites. Although the diffuse [M - 3H](3-) ions may show more HD scrambling, it is not possible to clearly distinguish HD scrambling from the expected deuterium uptake based on a hydrogen accessibility model. The capability of the IMS-HDX-MS/MS approach to provide relevant details about ion structure is discussed. Additionally, the ability to extend the approach for locating protonation sites on positively-charged ions is presented.

Ion Mobility Spectrometry-Hydrogen Deuterium Exchange Mass Spectrometry of Anions: Part 3. Estimating Surface Area Exposure by Deuterium Uptake.

Gas-phase hydrogen deuterium exchange (HDX), collision cross section (CCS) measurement, and molecular dynamics simulation (MDS) techniques were utilized to develop and compare three methods for estimating the relative surface area exposure of separate peptide chains within bovine insulin ions. Electrosprayed [M - 3H](3-) and [M - 5H](5-) insulin ions produced a single conformer type with respective collision cross sections of 528 ± 5 Å(2) and 808 ± 2 Å(2). [M - 4H](4-) ions were comprised of more compact (Ω = 676 ± 3 Å(2)) and diffuse (i.e., more elongated, Ω = 779 ± 3 Å(2)) ion conformer types. Ions were subjected to HDX in the drift tube using D2O as the reagent gas. Collision-induced dissociation was used to fragment mobility-selected, isotopically labeled [M - 4H](4-) and [M - 5H](5-) ions into the protein subchains. Deuterium uptake levels of each chain can be explained by limited inter-chain isotopic scrambling upon collisional activation. Using nominal ion structures from MDS and a hydrogen accessibility model, the deuterium uptake for each chain was correlated to its exposed surface area. In separate experiments, the per-residue deuterium content for the protonated and deprotonated ions of the synthetic peptide KKDDDDDIIKIIK were compared. The differences in deuterium content indicated the regional HDX accessibility for cations versus anions. Using ions of similar conformational type, this comparison highlights the complementary nature of HDX data obtained from positive- and negative-ion analysis.

Gas-Phase Hydrogen-Deuterium Exchange Labeling of Select Peptide Ion Conformer Types: a Per-Residue Kinetics Analysis.

The per-residue, gas-phase hydrogen deuterium exchange (HDX) kinetics for individual amino acid residues on selected ion conformer types of the model peptide KKDDDDDIIKIIK have been examined using ion mobility spectrometry (IMS) and HDX-tandem mass spectrometry (MS/MS) techniques. The [M + 4H](4+) ions exhibit two major conformer types with collision cross sections of 418 Å(2) and 446 Å(2); the [M + 3H](3+) ions also yield two different conformer types having collision cross sections of 340 Å(2) and 367 Å(2). Kinetics plots of HDX for individual amino acid residues reveal fast- and slow-exchanging hydrogens. The contributions of each amino acid residue to the overall conformer type rate constant have been estimated. For this peptide, N- and C-terminal K residues exhibit the greatest contributions for all ion conformer types. Interior D and I residues show decreased contributions. Several charge state trends are observed. On average, the D residues of the [M + 3H](3+) ions show faster HDX rate contributions compared with [M + 4H](4+) ions. In contrast the interior I8 and I9 residues show increased accessibility to exchange for the more elongated [M + 4H](4+) ion conformer type. The contribution of each residue to the overall uptake rate showed a good correlation with a residue hydrogen accessibility score model calculated using a distance from charge site and initial incorporation site for nominal structures obtained from molecular dynamic simulations (MDS).

Ion mobility spectrometry-hydrogen deuterium exchange mass spectrometry of anions: part 1. Peptides to proteins.

Ion mobility spectrometry (IMS) coupled with hydrogen deuterium exchange (HDX)-mass spectrometry (MS) has been used to study the conformations of negatively-charged peptide and protein ions. Results are presented for ion conformers of angiotensin 1, a synthetic peptide (SP), bovine insulin, ubiquitin, and equine cytochrome c. In general, the SP ion conformers demonstrate a greater level of HDX efficiency as a greater proportion of the sites undergo HDX. Additionally, these ions exhibit the fastest rates of exchange. Comparatively, the angiotensin 1 ions exhibit a lower rate of exchange and HDX level presumably because of decreased accessibility of exchange sites by charge sites. The latter are likely confined to the peptide termini. Insulin ions show dramatically reduced HDX levels and exchange rates, which can be attributed to decreased conformational flexibility resulting from the disulfide bonds. For the larger ubiquitin and protein ions, increased HDX is observed for larger ions of higher charge state. For ubiquitin, a conformational transition from compact to more elongated species (from lower to higher charge states) is reflected by an increase in HDX levels. These results can be explained by a combination of interior site protection by compact conformers as well as decreased access by charge sites. The elongated cytochrome c ions provide the largest HDX levels where higher values correlate with charge state. These results are consistent with increased exchange site accessibility by additional charge sites. The data from these enhanced IMS-HDX experiments are described in terms of charge site location, conformer rigidity, and interior site protection.

Combining ion mobility spectrometry with hydrogen-deuterium exchange and top-down MS for peptide ion structure analysis.

The gas-phase conformations of electrosprayed ions of the model peptide KKDDDDIIKIIK have been examined by ion mobility spectrometry (IMS) and hydrogen deuterium exchange (HDX)-tandem mass spectrometry (MS/MS) techniques. [M+4H](4+) ions exhibit two conformers with collision cross sections of 418 Å(2) and 471 Å(2). [M+3H](3+) ions exhibit a predominant conformer with a collision cross section of 340 Å(2) as well as an unresolved conformer (shoulder) with a collision cross section of ~367 Å(2). Maximum HDX levels for the more compact [M+4H](4+) ions and the compact and partially-folded [M+3H](3+) ions are ~12.9, ~15.5, and ~14.9, respectively. Ion structures obtained from molecular dynamics simulations (MDS) suggest that this ordering of HDX level results from increased charge-site/exchange-site density for the more compact ions of lower charge. Additionally, a new model that includes two distance calculations (charge site to carbonyl group and carbonyl group to exchange site) for the computer-generated structures is shown to better correlate to the experimentally determined per-residue deuterium uptake. Future comparisons of IMS-HDX-MS data with structures obtained from MDS are discussed with respect to novel experiments that will reveal the HDX rates of individual residues.

A new ion mobility-linear ion trap instrument for complex mixture analysis.

A new instrument that couples a low-pressure drift tube with a linear ion trap mass spectrometer is demonstrated for complex mixture analysis. The combination of the low-pressure separation with the ion trapping capabilities provides several benefits for complex mixture analysis. These include high sensitivity, unique ion fragmentation capabilities, and high reproducibility. Even though the gas-phase separation and the mass measurement steps are each conducted in an ion filtering mode, detection limits for mobility-selected peptide ions are in the tens of attomole range. In addition to ion separation, the low-pressure drift tube can be used as an ion fragmentation cell yielding mobility-resolved fragment ions that can be subsequently analyzed by multistage tandem mass spectrometry (MS(n)) methods in the ion trap. Because of the ion trap configuration, these methods can be comprised of any number (limited by ion signal) of collision-induced dissociation (CID) and electron transfer dissociation (ETD) processes. The high reproducibility of the gas-phase separation allows for comparison of two-dimensional ion mobility spectrometry (IMS)-MS data sets in a pixel-by-pixel fashion without the need for data set alignment. These advantages are presented in model analyses representing mixtures encountered in proteomics and metabolomics experiments.