Digital Quadrupole Isolation and Electron Capture Dissociation on an Extended Mass Range Q-TOF Provides Sequence and Structure Information on Proteins and Protein Complexes.

Electron capture dissociation (ECD) is now a well-established method for sequencing peptides and performing top-down analysis on proteins of less than 30 kDa, and there is growing interest in using this approach for studies of larger proteins and protein complexes. Although much progress on ECD has been made over the past few decades, establishing methods for obtaining informative spectra still poses a significant challenge. Here we describe how digital quadrupole (DigiQ) ion isolation can be used for the mass selection of single charge states of proteins and protein complexes prior to undergoing ECD and/or charge reduction. First, we demonstrate that the DigiQ can isolate single charge states of monomeric proteins such as ubiquitin (8.6 kDa) and charge states of large protein complexes such as pyruvate kinase (234 kDa) using a hybrid quadrupole-TOF-MS (Agilent extended m/z range 6545XT). Next, we demonstrate that fragment ions resulting from ECD can be utilized to provide information about the sequence and structure of the cytochrome c/heme complex and the ubiquitin monomer. Lastly, an especially interesting result for DigiQ isolation and electron capture (EC) was noted; namely, the 16+ charge state of the streptavidin/biotin complex reveals different electron capture patterns for the biotinylated proteoforms of streptavidin. This result is consistent with previous reports that apo streptavidin exists in multiple conformations and that biotin binding shifts the conformational dynamics of the complex (Quintyn, R. Chem. Biol. 2015, 22 (55), 583-592).

Search for Br* production in the D+DBr reaction.

Deuterium bromide (DBr) is expanded from a pulsed jet into a vacuum and a synchronized pulsed laser causes photodissociation of some of the DBr molecules to produce primarily (approximately 85%) ground-state bromine atoms ((2)P(3/2)) and fast D atoms. The latter collide with the cold DBr molecules and react to produce molecular deuterium (D(2)) via two possible channels, the adiabatic channel D(2)+Br((2)P(3/2)) and the nonadiabatic channel D(2)+Br*((2)P(1/2)), which are asymptotically separated in energy by the spin-orbit splitting (0.457 eV) of the bromine atom. Ion images are recorded for D(2)(v'=1, J'=16, 18-21), D(2)(v'=2, J'=6,7, 10-12, 14-16), and D(2)(v'=3, J'=2-5) for various collision energies. For the nonadiabatic production of spin-orbit-excited Br* in the D+DBr reaction for the conditions studied we estimate that this channel contributes 1% or less.

Vibrationally inelastic H + D2 collisions are forward-scattered.

We have measured differential cross sections (DCSs) for the vibrationally inelastic scattering process H + o-D(2)(v = 0, j = 0,2) --> H + o-D(2)(v' = 1-4, j' even). Several different collision energies and nearly the entire range of populated product quantum states are studied. The products are dominantly forward-scattered in all cases. This behavior is the opposite of what is predicted by the conventional textbook mechanism, in which collisions at small impact parameters compress the bond and cause the products to recoil in the backward direction. Recent quasiclassical trajectory (QCT) calculations examining only the o-D(2)(v' = 3, j') products suggest that vibrationally inelastic scattering is the result of a frustrated reaction in which the D-D bond is stretched, but not broken, during the collision. These QCT calculations provide a qualitative explanation for the observed forward-scattering, but they do not agree with experiments at the lowest values of j'. The present work shows that quantum mechanical calculations agree closely with experiments and expands upon previous results to show that forward-scattering is universally observed in vibrationally inelastic H + D(2) collisions over a broad range of conditions.

Vibrational excitation through tug-of-war inelastic collisions.

Vibrationally inelastic scattering is a fundamental collision process that converts some of the kinetic energy of the colliding partners into vibrational excitation(,). The conventional wisdom is that collisions with high impact parameters (where the partners only 'graze' each other) are forward scattered and essentially elastic, whereas collisions with low impact parameters transfer a large amount of energy into vibrations and are mainly back scattered. Here we report experimental observations of exactly the opposite behaviour for the simplest and most studied of all neutral-neutral collisions: we find that the inelastic scattering process H + D(2)(v = 0, j = 0, 2) --> H + D(2)(v' = 3, j' = 0, 2, 4, 6, 8) leads dominantly to forward scattering (v and j respectively refer to the vibrational and rotational quantum numbers of the D(2) molecule). Quasi-classical trajectory calculations show that the vibrational excitation is caused by extension, not compression, of the D-D bond through interaction with the passing H atom. However, the H-D interaction never becomes strong enough for capture of the H atom before it departs with diminished kinetic energy; that is, the inelastic scattering process is essentially a frustrated reaction in which the collision typically excites the outward-going half of the H-D-D symmetric stretch before the H-D(2) complex dissociates. We suggest that this 'tug of war' between H and D(2) is a new mechanism for vibrational excitation that should play a role in all neutral-neutral collisions where strong attraction can develop between the collision partners.

Corroboration of theory for H + D2 --> D + HD (v' = 3, j' = 0) reactive scattering dynamics.

The differential cross section (DCS) for the reaction H + D2 --> D + HD (v' = 3, j' = 0) exhibits particularly rich dynamics; in addition to the expected direct recoil backscattering feature, a surprising time-delayed forward scattering feature appears that has been attributed to glory scattering arising from nearside and farside interference. This fact leads to a complex DCS that depends strongly on the collision energy. Its accurate calculation requires a fully quantum mechanical (QM) treatment. We report improved measurements of this DCS over the collision energy range 1.55 < or = E(coll) < or = 1.82 eV. Previous measurements using the core extraction method, while generally in agreement with theory, lacked sufficient resolution to capture all of the noteworthy behavior of the system; in the present work, we use ion imaging to observe many previously unresolved features of the DCS, particularly in the forward-scattered region. Agreement with QM calculations is found at all collision energies, reconciling an earlier discrepancy between experiment and theory near E(coll) = 1.54 eV.

Differential cross section for the H+D(2)-->HD(v(')=1,j(')=2,6,10)+D reaction as a function of collision energy.

We have measured differential cross sections (DCSs) for the HD (v(')=1,j(')=2,6,10) products of the H+D(2) exchange reaction at five different collision energies in the range 1.48< or =E(coll)< or =1.94 eV. The contribution from the less energetic H atoms formed upon spin-orbit excitation of Br in the photolysis of the HBr precursor is taken into account for two collision energies, E(coll)=1.84 and 1.94 eV, allowing us to disentangle the two different channels. The measured DCSs agree well with new time-dependent quantum-mechanical calculations. As the product rotational excitation increases, the DCSs shift from backward to sideward scattering, as expected. We also find that the shapes of the DCSs show only a small overall dependence on the collision energy, with a notable exception occurring for HD (v(')=1,j(')=2), which appears bimodal at high collision energies. We suggest that this feature results from both direct recoil and indirect scattering from the conical intersection.

Construction and calibration of an instrument for three-dimensional ion imaging.

We describe a new instrument based on a delay-line detector for imaging the complete three-dimensional velocity distribution of photoionized products from photoinitiated reactions. Doppler-free [2+1] resonantly enhanced multiphoton ionization (REMPI) of H and D atoms formed upon photolysis of HBr and DBr in the range 203 nm < or = lambda photolysis < or = 243 nm yields radial speeds measured to be accurate within 1% of those calculated. The relative speed resolution is about 5% and limited by photoionization recoil broadening. A relative speed resolution of 3.4% is obtained for [3+1] REMPI, which minimizes the ionization recoil. We also determine the branching ratio between ground-state and spin-orbit-excited product channels and their associated anisotropies. We find that DBr photolysis dynamics differs slightly from its HBr counterpart.

Collision-energy dependence of HD(nu'=1,j') product rotational distributions for the H + D2 reaction.

Product rotational distributions for the reaction H + D2 --> HD(nu'=1,j') + D have been measured for 16 collision energies in the range of 1.43 < or = E(coll) < or = 2.55 eV. Time-dependent quantum-mechanical calculations agree well in general with the experimental results, but they consistently yield slightly colder distributions. In terms of the average energy channeled into rotation, the differences between experiment and theory amount to approximately 10% for all collision energies sampled. No peculiarity is found for E(coll)=2.55 eV at which the system has sufficient energy to access the first HD2 electronically excited state.