Surface-induced Dissociation Experiments Research Articles

Surface-Induced Dissociation for Protein Complex Characterization.

Native mass spectrometry (nMS) enables intact non-covalent complexes to be studied in the gas phase. nMS can provide information on composition, stoichiometry, topology, and, when coupled with surface-induced dissociation (SID), subunit connectivity. Here we describe the characterization of protein complexes by nMS and SID. Substructural information obtained using this method is consistent with the solved complex structure, when a structure exists. This provides confidence that the method can also be used to obtain substructural information for unknowns, providing insight into subunit connectivity and arrangements. High-energy SID can also provide information on proteoforms present. Previously SID has been limited to a few in-house modified instruments and here we focus on SID implemented within an in-house-modified Q Exactive UHMR. However, SID is currently commercially available within the Waters Select Series Cyclic IMS instrument. Projects are underway that involve the NIH-funded native MS resource (nativems.osu.edu), instrument vendors, and third-party vendors, with the hope of bringing the technology to more platforms and labs in the near future. Currently, nMS resource staff can perform SID experiments for interested research groups.

Prediction of Protein Complex Structure Using Surface-Induced Dissociation and Cryo-Electron Microscopy.

A variety of techniques involving the use of mass spectrometry (MS) have been developed to obtain structural information on proteins and protein complexes. One example of these techniques, surface-induced dissociation (SID), has been used to study the oligomeric state and connectivity of protein complexes. Recently, we demonstrated that appearance energies (AE) could be extracted from SID experiments and that they correlate with structural features of specific protein-protein interfaces. While SID AE provides some structural information, the AE data alone are not sufficient to determine the structures of the complexes. For this reason, we sought to supplement the data with computational modeling, through protein-protein docking. In a previous study, we demonstrated that the scoring of structures generated from protein-protein docking could be improved with the inclusion of SID data; however, this work relied on knowledge of the correct tertiary structure and only built full complexes for a few cases. Here, we performed docking using input structures that require less prior knowledge, using homology models, unbound crystal structures, and bound+perturbed crystal structures. Using flexible ensemble docking (to build primarily subcomplexes from an ensemble of backbone structures), the RMSD100 of all (15/15) predicted structures using the combined Rosetta, cryo-electron microscopy (cryo-EM), and SID score was less than 4 Å, compared to only 7/15 without SID and cryo-EM. Symmetric docking (which used symmetry to build full complexes) resulted in predicted structures with RMSD100 less than 4 Å for 14/15 cases with experimental data, compared to only 5/15 without SID and cryo-EM. Finally, we also developed a confidence metric for which all (26/26) proteins flagged as high confidence were accurately predicted.

Surface-induced dissociation: a unique tool for studying energetics and kinetics of the gas-phase fragmentation of large ions.

Surface-induced dissociation (SID) is a valuable tool for investigating the activation and dissociation of large ions in tandem mass spectrometry. This account summarizes key findings from studies of the energetics and mechanisms of complex ion dissociation in which SID experiments were combined with Rice-Ramsperger-Kassel-Marcus modeling of the experimental data. These studies used time- and collision-energy-resolved SID experiments and SID combined with resonant ejection of selected fragment ions on a specially designed Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Fast-ion activation by collision with a surface combined with the long and variable timescale of FT-ICR mass spectrometry is perfectly suited to studying the energetics and dynamics of complex ion dissociation in the gas phase. Modeling of time- and collision-energy-resolved SID enables the accurate determination of energy and entropy effects in the dissociation process. It has been demonstrated that entropy effects play an important role in determining the dissociation rates of both covalent and noncovalent bonds in large gaseous ions. SID studies have provided important insights on the competition between charge-directed and charge-remote fragmentation in even-electron peptide ions and the role of the charge and radical site on the energetics of the dissociation of odd-electron peptide ions. Furthermore, this work examined factors that affect the strength of noncovalent binding, as well as the competition between covalent and noncovalent bond cleavages and between proton and electron transfer in model systems. Finally, SID studies have been used to understand the factors affecting nucleation and growth of clusters in solution and in the gas phase.

Effect of the basic residue on the energetics and dynamics of dissociation of phosphopeptides

Time- and collision-energy-resolved surface-induced dissociation (SID) of protonated peptides containing phosphoserine (s) was studied using a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer configured for SID experiments. We examined fragmentation of three singly protonated peptides: GGGsGGG, KGGsGGG and RGGsGGG. Fragmentation of GGGsGGG occurs under the mobile proton condition, while the ionizing proton is sequestered by the basic residue, resulting in the nonmobile proton condition in dissociation of the two basic peptides: KGGsGGG and RGGsGGG. RRKM modeling of the experimental data demonstrates that the energetics and dynamics of H3PO4 loss are different under mobile and nonmobile proton conditions. Specifically, fragmentation of GGGsGGG is characterized by a higher dissociation barrier, 1.68eV, and higher activation entropy, 11e.u. (e.u.=entropy unit), than fragmentation of the basic peptides. Similar threshold energies of 1.36eV and 1.40eV and activation entropies of −4.9e.u. and 0.3e.u. were obtained for KGGsGGG and RGGsGGG, respectively. We propose that the loss of H3PO4 from phosphoserine is a two-step process, in which the phosphate abstraction from the phosphorylated side chain is followed by dissociation of the ion–molecule complex.

Energy and entropy effects in dissociation of peptide radical anions

Time- and collision energy-resolved surface-induced dissociation (SID) of peptide radical anions was studied using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) configured for SID experiments. Peptide radical cations and anions were produced by gas-phase fragmentation of CoIII(salen)-peptide complexes. The effect of the charge, radical, and the presence of a basic residue on the energetics and dynamics of dissociation of peptide ions was examined using RVYIHPF (1) and HVYIHPF (2) as model systems. Comparison of the survival curves of [M+H]+, [M−H]−, M+, and [M−2H]− ions of these precursors demonstrated that even-electron ions are more stable toward fragmentation than their odd-electron counterparts. RRKM modeling of the experimental data demonstrated that the lower stability of the positive radicals is mainly attributed to lower dissociation thresholds while entropy effects are responsible the relative instability of the negative radicals. Substitution of arginine with less basic histidine residue has a strong destabilizing effect on the [M+H]+ ions and a measurable stabilizing effect on the odd-electron ions. Lower threshold energies for dissociation of both positive and negative radicals of 1 are attributed to the presence of lower-energy dissociation pathways that are most likely promoted by the presence of arginine.

Effect of the Basic Residue on the Energetics, Dynamics, and Mechanisms of Gas-Phase Fragmentation of Protonated Peptides

The effect of the basic residue on the energetics, dynamics, and mechanisms of backbone fragmentation of protonated peptides was investigated. Time-resolved and collision energy-resolved surface-induced dissociation (SID) of singly protonated peptides with the N-terminal arginine residue and their analogues, in which arginine is replaced with less basic lysine and histidine residues, was examined using a specially configured Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). SID experiments demonstrated different kinetics of formation of several primary product ions of peptides with and without arginine residue. The energetics and dynamics of these pathways were determined from Rice-Ramsperger-Kassel-Marcus (RRKM) modeling of the experimental data. Comparison between the kinetics and energetics of fragmentation of arginine-containing peptides and the corresponding methyl ester derivatives provides important information on the effect of dissociation pathways involving salt bridge (SB) intermediates on the observed fragmentation behavior. Because pathways involving SB intermediates are characterized by low threshold energies, they efficiently compete with classical oxazolone and imine/enol pathways of arginine-containing peptides on a long time scale of the FTICR instrument. In contrast, fragmentation of histidine- and lysine-containing peptides is largely determined by canonical pathways. Because SB pathways are characterized by negative activation entropies, fragmentation of arginine-containing peptides is kinetically hindered and observed at higher collision energies as compared to their lysine- and histidine-containing analogues.

The Effect of the Secondary Structure on Dissociation of Peptide Radical Cations: Fragmentation of Angiotensin III and Its Analogues

Fragmentation of protonated RVYIHPF and RVYIHPF-OMe and the corresponding radical cations was studied using time- and collision energy-resolved surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Peptide radical cations were produced by gas-phase fragmentation of Co (III)(salen)-peptide complexes. Both the energetics and the mechanisms of dissociation of even-electron and odd-electron angiotensin III ions are quite different. Protonated molecules are much more stable toward fragmentation than the corresponding radical cations. RRKM modeling of the experimental data suggests that this stability is largely attributed to differences in threshold energies for dissociation, while activation entropies are very similar. Detailed analysis of the experimental data obtained for radical cations demonstrated the presence of two distinct structures separated by a high free-energy barrier. The two families of structures were ascribed to the canonical and zwitterionic forms of the radical cations produced in our experiments.

Experimental and Theoretical Studies of the Structures and Interactions of Vancomycin Antibiotics with Cell Wall Analogues

Surface-induced dissociation (SID) of the singly protonated complex of vancomycin antibiotic with cell wall peptide analogue (N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-Ala) was studied using a 6 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR MS) specially configured for SID experiments. The binding energy between the vancomycin and the peptide was obtained from the RRKM modeling of the time- and energy-resolved fragmentation efficiency curves (TFECs) of the precursor ion and its fragments. Molecular dynamics simulations of the vancomycin, peptide, and vancomycin-peptide complex were carried out to explore the low energy conformations. Density functional theory (DFT) calculations of the geometries, proton affinities, and binding energies were performed for several model systems including vancomycin (V), vancomycin aglycon (VA), N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-Ala, and noncovalent complexes of VA with N-acetyl-D-Ala-D-Ala and V with N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-Ala. Comparison between the experimental and computational results suggests that the most probable structure of the complex observed in our experiments corresponds to the neutral peptide bound to the vancomycin protonated at the primary amine of the disaccharide group. The experimental binding energy of 30.9 +/- 1.8 kcal/mol is in good agreement with the binding energy of 36.3-42.0 kcal/mol calculated for the model system representing the preferred structure of the complex.

Energetics and Dynamics of Electron Transfer and Proton Transfer in Dissociation of MetalIII(salen)−Peptide Complexes in the Gas Phase

Time- and collision energy-resolved surface-induced dissociation (SID) of ternary complexes of Co(III)(salen)+, Fe(III)(salen)+, and Mn(III)(salen)+ with several angiotensin peptide analogues was studied using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Time-resolved fragmentation efficiency curves (TFECs) were modeled using an RRKM-based approach developed in our laboratory. The approach utilizes a very flexible analytical expression for the internal energy deposition function that is capable of reproducing both single-collision and multiple-collision activation in the gas phase and excitation by collisions with a surface. The energetics and dynamics of competing dissociation pathways obtained from the modeling provides important insight on the competition between proton transfer, electron transfer, loss of neutral peptide ligand, and other processes that determine gas-phase fragmentation of these model systems. Similar fragmentation behavior was obtained for various Co(III)(salen)-peptide systems of different angiotensin analogues. In contrast, dissociation pathways and relative stabilities of the complexes changed dramatically when cobalt was replaced with trivalent iron or manganese. We demonstrate that the electron-transfer efficiency is correlated with redox properties of the metal(III)(salen) complexes (Co > Fe > Mn), while differences in the types of fragments formed from the complexes reflect differences in the modes of binding between the metal-salen complex and the peptide ligand. RRKM modeling of time- and collision-energy-resolved SID data suggests that the competition between proton transfer and electron transfer during dissociation of Co(III)(salen)-peptide complexes is mainly determined by differences in entropy effects while the energetics of these two pathways are very similar.

Fragmentation energetics for angiotensin II and its analogs from time- and energy-resolved surface-induced dissociation studies

Surface-induced dissociation (SID) of four model peptides: DRVYIHPF, RVYIHPF, RVYIHAF, and RVYIHDF was studied using a novel Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially configured for SID experiments. The energetics and dynamics of peptide fragmentation were deduced by modeling the time- and energy-resolved survival curves for each precursor ion using an RRKM based approach developed in our laboratory. Accurate dissociation parameters can be obtained from these experiments because collision-energy-resolved SID data are very sensitive to both the energetics and dynamics of dissociation. We found that transition from selective to non-selective fragmentation as ion kinetic energy is increased is associated with a substantial (ca. 0.5 eV) increase in the dissociation energy and a 3–4 orders of magnitude increase in the pre-exponential factor. Dissociation thresholds for angiotensin analogs derived from the experimental data are as follows: 1.62 eV for RVYIHAF and RVYIHPF, 1.14 eV for RVYIHDF and 1.13 eV for DRVYIHPF. Pre-exponential factors of 8.2×10 11, 7.2×10 12, 3.1×10 8, and 5.0×10 7 s −1 were obtained for RVYIHPF, RVYIHAF, RVYIHDF, and DRVYIHPF, respectively. Contribution from shattering to the total decomposition of the precursor ion increases for kinetically hindered fragmentation. The largest contribution is observed for a peptide ion that has the largest negative reaction entropy—DRVYIHPF.

Shattering of Peptide ions on self-assembled monolayer surfaces.

Time- and collision energy-resolved surface-induced dissociation (SID) of des-Arg(1)- and des-Arg(9)-bradykinin on a fluorinated self-assembled monolayer (SAM) surface was studied by use of a novel Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Time-resolved fragmentation efficiency curves (TFECs) were modeled by an RRKM-based approach developed in our laboratory that utilizes a very flexible analytical expression for the internal energy deposition function capable of reproducing both single- and multiple-collision activation in the gas phase and excitation by collisions with a surface. Both experimental observations and modeling establish a very sharp transition in the dynamics of ion-surface interaction: the shattering transition. The experimental signature for this transition is the appearance of prompt (time-independent) fragmentation, which becomes dominant at high collision energies. Shattering opens a variety of dissociation pathways that are not accessible to slow collisional and thermal ion activation. This results in much better sequence coverage for the singly protonated peptides than dissociation patterns obtained with any of the slow activation methods. Modeling demonstrated that, for short reaction delays, dissociation of these peptides is solely determined by shattering. Internal energies required for shattering transition are approximately the same for des-Arg(1) and des-Arg(9)-bradykinin, resulting in the overlap of fragmentation efficiency curves obtained at short reaction delays. At longer delay times, parent ions depletion is mainly determined by a slow decay rate and fragmentation efficiency curves for des-Arg(1) and des-Arg(9)-bradykinin diverge. Dissociation thresholds of 1.17 and 1.09 eV and activation entropies of -22.2 and -23.3 cal/(mol K) were obtained for des-Arg(1) and des-Arg(9)-bradykinin from RRKM modeling of time-resolved data. Dissociation parameters for des-Arg(1)-bradykinin are in good agreement with parameters derived from thermal experiments. However, there is a significant discrepancy between the thermal data and dissociation parameters for des-Arg(9)-bradykinin obtained in this study. The difference is attributed to the differences in conformations that undergo thermal activation and activation by ion-surface collisions prior to dissociation.

Energetics of selective cleavage at acidic residues studied by time- and energy-resolved surface-induced dissociation in FT-ICR MS

Surface-induced dissociation (SID) of four model peptides: LDIFSDF, LDIFSDFR, RLDIFSDF, and LEIFSEFR, was studied using a novel Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. The energetics and dynamics of selective cleavages at acidic residues were deduced by modeling the time- and energy-resolved fragmentation efficiency curves (TFECs) using an RRKM-based approach developed in our laboratory. RRKM modeling revealed that addition of a basic residue—arginine—to the C-terminus of a peptide has a very small effect on the dissociation threshold. However, the dynamics of dissociation is dramatically affected by the presence of arginine. The Arrhenius pre-exponential factor for dissociation of LDIFSDF is two orders of magnitude higher than the pre-exponential factor for dissociation of arginine-containing peptides. The difference in the pre-exponential factors is indicative of a complex rearrangement process associated with selective fragmentation. Molecular mechanics modeling of the four parent ions gives some qualitative insight into the differences in fragmentation mechanisms.

Optimization of a matrix-assisted laser desorption ionization-ion mobility-surface-induced dissociation-orthogonal-time-of-flight mass spectrometer: simultaneous acquisition of multiple correlated MS1 and MS2 spectra

Optimization of a matrix-assisted laser desorption ionization-ion mobility-surface-induced dissociation-o-time-of-flight mass spectrometer for peptide sequencing is discussed. Surface-induced dissociation (SID) spectra obtained by using stainless steel, Au grids, and fluorinated self-assembled monolayers (F-SAM) on Au are compared. The F-SAM surfaces yield similar fragment ions to those obtained using an adventitious hydrocarbon coated stainless steel surface; however, optimum collision energies differ for the two surfaces. The advantage of ion mobility-time-of-flight (TOF) is the ability to simultaneously acquire MS1 and MS2 spectra, which greatly facilitates high-throughput sequencing of peptides and mixtures of peptides resulting form the proteolytic digest of proteins. Simultaneous acquisition of ion mobility and TOF spectra introduces a time element to SID experiments that can be used as a probe of ion/surface interactions.