An Orbitrap/Time-of-Flight Mass Spectrometer for Photofragment Ion Imaging and High-Resolution Mass Analysis of Native Macromolecular Assemblies.

We discuss the design, development, and evaluation of an Orbitrap/time-of-flight (TOF) mass spectrometry (MS)-based instrument with integrated UV photodissociation (UVPD) and time/mass-to-charge ratio (m/z)-resolved imaging for the comprehensive study of the higher-order molecular structure of macromolecular assemblies (MMAs). A bespoke TOF analyzer has been coupled to the higher-energy collisional dissociation cell of an ultrahigh mass range hybrid quadrupole-Orbitrap MS. A 193 nm excimer laser was employed to photofragment MMA ions. A combination of microchannel plates (MCPs)-Timepix (TPX) quad and MCPs-phosphor screen-TPX3CAM assemblies have been used as axial and orthogonal imaging detectors, respectively. The instrument can operate in four different modes, where the UVPD-generated fragment ions from the native MMA ions can be measured with high-mass resolution or imaged in a mass-resolved manner to reveal the relative positions of the UVPD fragments postdissociation. This information is intended to be utilized for retrieving higher-order molecular structural details that include the conformation, subunit stoichiometry, and molecular interactions as well as to understand the dissociation dynamics of the MMAs in the gas phase.

Coherent diffractive imaging of proteins and viral capsids: simulating MS SPIDOC.

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm-1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

The Omnitrap Platform: A Versatile Segmented Linear Ion Trap for Multidimensional Multiple-Stage Tandem Mass Spectrometry.

Multidimensional multiple-stage tandem processing of ions is demonstrated successfully in a novel segmented linear ion trap. The enhanced performance is enabled by incorporating the entire range of ion activation methods into a single platform in a highly dynamic fashion. The ion activation network comprises external injection of reagent ions, radical neutral species, photons, electrons, and collisions with neutrals. Axial segmentation of the two-dimensional trapping field provides access to a unique functionality landscape through a system of purpose-designed regions for processing ions with maximum flexibility. Design aspects of the segmented linear ion trap, termed the Omnitrap platform, are highlighted, and motion of ions trapped by rectangular waveforms is investigated experimentally by mapping the stability diagram, tracing secular frequencies, and exploring different isolation techniques. All fragmentation methods incorporated in the Omnitrap platform involving radical chemistry are shown to provide complete sequence coverage for partially unfolded ubiquitin. Three-stage (MS3) tandem mass spectrometry experiments combining collision-induced dissociation of radical ions produced by electron meta-ionization and further involving two intermediate steps of ion isolation and accumulation are performed with high efficiency, producing information rich spectra with signal-to-noise levels comparable to those obtained in a two-stage (MS2) experiment. The advanced capabilities of the Omnitrap platform to provide in-depth top-down MSn characterization of proteins are portrayed. Performance is further enhanced by connecting the Omnitrap platform to an Orbitrap mass analyzer, while successful integration with time-of-flight analyzers has already been demonstrated.