Dmitry Grinfeld

Defining the Stoichiometry and Cargo Load of Viral and Bacterial Nanoparticles by Orbitrap Mass Spectrometry

Accurate mass analysis can provide useful information on the stoichiometry and composition of protein-based particles, such as virus-like assemblies. For applications in nanotechnology and medicine, such nanoparticles are loaded with foreign cargos, making accurate mass information essential to define the cargo load. Here, we describe modifications to an Orbitrap mass spectrometer that enable high mass analysis of several virus-like nanoparticles up to 4.5 MDa in mass. This allows the accurate determination of the composition of virus-like particles. The modified instrument is utilized to determine the cargo load of bacterial encapsulin nanoparticles that were engineered to encapsulate foreign cargo proteins. We find that encapsulin packages from 8 up to 12 cargo proteins, thereby quantifying cargo load but also showing the ensemble spread. In addition, we determined the previously unknown stoichiometry of the three different splice variants of the capsid protein in adeno-associated virus (AAV) capsids, showing that symmetry is broken and assembly is heterogeneous and stochastic. These results demonstrate the potential of high-resolution mass analysis of protein-based nanoparticles, with widespread applications in chemical biology and nanotechnology.

The 3D OrbiSIMS—label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power

We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 μm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters—gamma-aminobutyric acid, dopamine and serotonin—with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.

Space-charge effects in an electrostatic multireflection ion trap.

The multireflection ion traps with isochronous properties offer a lot of opportunities for time-of-flight mass spectrometry by elongation of the ion path, thus preserving the compact dimensions of an instrument. We have built and tested a two-mirror linear trap that provides at least 80,000 mass-resolving power. Although the mass resolution appears promising, there are substantial limitations that arise from Coulomb interactions of the trapped ions. Among these, the mutual repulsion of ions with same or close mass-to-charge ratios appears dominant, resulting in counterintuitive motion synchronization. The self-bunching and coalescence effects are also examined by numerical simulation.

Phase-Constrained Spectrum Deconvolution for Fourier Transform Mass Spectrometry

This Article introduces a new computationally efficient noise-tolerant signal processing method, referred to as phased spectrum deconvolution method (ΦSDM), designed for Fourier transform mass spectrometry (FT MS). ΦSDM produces interference-free mass spectra with resolution beyond the Fourier transform (FT) uncertainty limit. With a presumption that the oscillation phases are preserved, the method deconvolves an observed FT spectrum into a distribution of harmonic components bound to a fixed frequency grid, which is several times finer than that of FT. The approach shows stability under noisy conditions, and the noise levels in the resulting spectra are lower than those of the original FT spectra. Although requiring more computational power than standard FT algorithms, ΦSDM runs in a quasilinear time. The method was tested on both synthetic and experimental data, and consistently demonstrated performance superior to the FT-based methodologies, be it across the entire mass range or on a selected mass window of interest. ΦSDM promises substantial improvements in the spectral quality and the speed of FT MS instruments. It might also be beneficial for other spectroscopy approaches which require harmonic analysis for data processing.

Some aspects of space-charge effect calculation in high-resolution mass spectrometry

A variational 3D approach to the problem of simulating stationary distributions of ions in the radiofrequency low-vacuum ion traps with regard to Coulomb interaction and collisions of ions with buffer gas molecules is proposed. The software developed in the course of this work is employed to study the structure of stationary ion ensembles in the radiofrequency ion traps of various types. The effect of high-frequency and constant voltages, space-charge density, and buffer gas temperature on the formation of stationary distributions in the radiofrequency ion traps and their limiting capacitance is investigated. It is shown that the use of electrodes with a constant voltage in the presence of high enough ion density allows pre-filtering of ions directly in a high-frequency trap-accumulator.

Simulation of the stationary distributions of ions in radiofrequency low-vacuum traps with regard to the coulomb interaction

The paper presents main peculiarities of implementation and testing of an algorithm based on the variational approach to the problem of simulating the stationary distributions of ions in the radiofrequency, low-vacuum ion traps with taking into consideration the Coulomb interaction and interaction with buffer gas. A good agreement between the results of numerical modeling and analytical results obtained earlier by other authors for simpler models is attained. The employment of the software that has been developed in the course of this work enables studying the structure of ion ensembles in the radiofrequency ion traps of different types and obtaining the results being of interest for high-resolution mass spectrometry. The algorithm allows a natural generalization to three-dimensional case.

Determination of Collision Cross-Sections of Protein Ions in an Orbitrap Mass Analyzer

We demonstrate a method for determining the collision cross-sections (CCSs) of protein ions based on the decay rate of the time-domain transient signal from an Orbitrap mass analyzer. Multiply charged ions of ubiquitin, cytochrome c, and myoglobin were generated by electrospray ionization of both denaturing solutions and ones with high salt content to preserve native-like structures. A linear relationship between the pressure in the Orbitrap analyzer and the transient decay rate was established and used to demonstrate that the signal decay is primarily due to ion-neutral collisions for protein ions across the entire working pressure range of the instrument. The CCSs measured in this study were compared with previously published CCS values measured by ion mobility mass spectrometry (IMS), and results from the two methods were found to differ by less than 7% for all charge states known to adopt single gas-phase conformations.

Limits for resolving tandem mass tag reporter ions with identical integer mass using phase constrained spectrum deconvolution

A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on TMT reporter ion quantification with identical integer masses.

Limits for Resolving Isobaric Tandem Mass Tag Reporter Ions Using Phase-Constrained Spectrum Deconvolution

A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT), which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here, we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments, relying on isobaric TMT reporter ion quantification.