Structural studies of metal ligand complexes by ion mobility-mass spectrometry

The final publication is available at Springer via http://dx.doi.org/10.1007/s12127-013-0122-8Collision cross sections (CCS) have been measured for three salen ligands, and their complexes with copper and zinc using travelling-wave ion mobility-mass spectrometry (TWIMS) and drift tube ion mobility-mass spectrometry (DTIMS), allowing a comparative size evaluation of the ligands and complexes. CCS measurements using TWIMS were determined using peptide and TAAH calibration standards. TWIMS measurements gave significantly larger CCS than DTIMS in helium, by 9 % for TAAH standards and 3 % for peptide standards, indicating that the choice of calibration standards is important in ensuring the accuracy of TWIMS-derived CCS measurements. Repeatability data for TWIMS was obtained for inter- and intra-day studies with mean RSDs of 1. 1 % and 0. 7 %, respectively. The CCS data obtained from IM-MS measurements are compared to CCS values obtained via the projection approximation, the exact hard spheres method and the trajectory method from X-ray coordinates and modelled structures using density functional theory (DFT) based methods. © 2013 Springer-Verlag Berlin Heidelberg

Traveling-wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross-sections of human insulin oligomers.

RATIONALE: The collision cross-section (Ω) of a protein or protein complex ion can be measured using traveling-wave (T-wave) ion mobility (IM) mass spectrometry (MS) via calibration with compounds of known Ω. The T-wave Ω-values depend strongly on instrument parameters and calibrant selection. Optimization of instrument parameters and calibration standards are crucial for obtaining accurate T-wave Ω-values. METHODS: Human insulin and the fast-acting insulin aspart under native-like conditions (ammonium acetate, physiological pH) were analyzed on Waters SYNAPT G1 and G2 HDMS instruments. The calibrated T-wave Ω-values of insulin monomer, dimer, and hexamer ions were measured using many different combinations of denatured and native-like calibrants (masses between 2.85 and 256 kDa) and T-wave conditions. Drift-tube Ω-values were obtained on a modified SYNAPT G1. RESULTS: Insulin T-wave Ω-values were measured at 26 combinations of T-wave velocity and amplitude. Optimal sets of calibrants were identified that yield Ω-values with minimal dependence on T-wave conditions and calibration plots with high R(2)-values. The T-wave Ω-values determined under conditions satisfying these criteria had absolute errors <2%. Structural differences between human insulin and fast-acting insulin aspart were probed with IM-MS. Insulin aspart monomers have increased flexibility, while hexamers are more compact than human insulin. CONCLUSIONS: Accurate T-wave Ω-values that are indistinguishable from drift-tube values are obtained when using (1) native-like calibrants with masses that closely bracket that of the analyte, (2) T-wave velocities that maximize the R(2) of the calibration plot for those calibrants, and (3) at least three replicates at T-wave velocities that yield calibration plots with high R(2)

The interaction of the antitoxin DM43 with a snake venom metalloproteinase analyzed by mass spectrometry and surface plasmon resonance.

DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry (MS) and surface plasmon resonance (SPR) to improve the molecular characterization of this heterocomplex. The stoichiometry of the interaction was confirmed by nanoelectrospray ionization-quadrupole-time-of-flight MS; from native solution conditions, the complex showed a molecular mass of ~94 kDa, indicating that one molecule of jararhagin (50 kDa) interacts with one monomer of DM43 (43 kDa). Although readily observed in solution, the dimeric structure of the inhibitor was barely preserved in the gas phase. This result suggests that, in contrast to the toxin-antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real-time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti-jararhagin monoclonal antibody MAJar 2. The sensorgrams obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: k(a) = 3.54 ± 0.03 × 10(4) M(-1) s(-1) and k(d) = 1.16 ± 0.07 × 10(-5) s(-1), resulting in an equilibrium dissociation constant (K(D) ) of 0.33 ± 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with unequivocal implications for the use of this kind of molecule as template for the rational development of novel antivenom therapies

The Interaction of the Antitoxin DM43 with a Snake Venom Metalloproteinase Analyzed by Mass Spectrometry and Surface Plasmon Resonance

DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry (MS) and surface plasmon resonance (SPR) to improve the molecular characterization of this heterocomplex. The stoichiometry of the interaction was confirmed by nanoelectrospray ionization-quadrupole-time-of-flight MS; from native solution conditions, the complex showed a molecular mass of ~94 kDa, indicating that one molecule of jararhagin (50 kDa) interacts with one monomer of DM43 (43 kDa). Although readily observed in solution, the dimeric structure of the inhibitor was barely preserved in the gas phase. This result suggests that, in contrast to the toxin-antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real-time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti-jararhagin monoclonal antibody MAJar 2. The sensorgrams obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: k(a) = 3.54 ± 0.03 × 10(4) M(-1) s(-1) and k(d) = 1.16 ± 0.07 × 10(-5) s(-1), resulting in an equilibrium dissociation constant (K(D) ) of 0.33 ± 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with unequivocal implications for the use of this kind of molecule as template for the rational development of novel antivenom therapies

Collidoscope: An Improved Tool for Computing Collisional Cross-Sections with the Trajectory Method

Ion Mobility-Mass Spectrometry (IM-MS) can be a powerful tool for determining structural information about ions in the gas phase, from small covalent analytes to large, unfolded, and/or denatured proteins and complexes. For large biomolecular ions, which may have a wide variety of possible gas-phase conformations and multiple charge sites, quantitative, physically explicit modeling of collisional cross sections (CCSs) for comparison to IMS data can be challenging and time-consuming. We present a “trajectory method” (TM) based CCS calculator, named “Collidoscope”, which utilizes parallel processing and optimized trajectory sampling, and implements both He and N2 as collision gas options. Also included is a charge-placement algorithm for determining probable charge site configurations for protonated protein ions given an input geometry in pdb file format. Results from Collidoscope are compared to those from the current state-of-the-art CCS simulation suite, IMoS. Collidoscope CCSs are typically within 4% of IMoS values for ions with masses from ~18 Da to ~800 kDa. Collidoscope CCSs using x-ray crystal geometries are typically within a few percent of IM-MS experimental values for ions with mass up to ~3.5 kDa (melittin), and discrepancies for larger ions up to ~800 kDa (GroEL) are attributed in large part to changes in ion structure during and after the electrospray process. Due to its physically explicit modeling of scattering, computational efficiency, and accuracy, Collidoscope can be a valuable tool for IM-MS research, especially for large biomolecular ions