Tandem mass tags (TMT) allow highly multiplexed and thus high-throughput, precisely quantitative proteomic analysis. Incorporation of additional deuterated reporter channels has near-doubled the multiplexation achieved with Thermo Scientific™ TMTpro reagents from 18 to 35-plex but requires extremely high ~100k analyzer resolving power at m/z 128 to differentiate and quantify reporter ion channels, far beyond any single reflection time-of-flight analyzer, and exceeding the multi-reflection Thermo Scientific™ Astral™ analyzer in its standard operation. A multi-pass mode of Astral operation has been developed for the Thermo Scientific™ Orbitrap™ Astral™ Zoom mass spectrometer that triples the ion path to 90 m, more than doubling resolving power for a narrow m/z range. This TMT HR mode has been integrated into a new method of TMT proteomic analysis that splits regular MS2 analysis of labeled peptides into paired measurements comprising wide mass range scans for peptide identification, and TMT HR mode scans for reporter ion quantification. The method has been shown to accurately quantify 32-plex labeled HeLa protein lysate and provide far greater depth of analysis as state-of-the-art Orbitrap-only methods, while analysis of 11-plex labeled yeast showed no analytical depth sacrificed vs regular Orbitrap Astral TMT analysis. Further comparative measurements of a 2-cell line 35-plex sample demonstrated greater analytical depth, and similar quantitative precision, to gold standard Orbitrap MS3 methods.

Reflectron-based time-of-flight analyzers rely on sub-nanosecond detector time response to achieve acceptable resolving power for low-mid mass, multiple ion peaks. With the adoption of multi-reflection analyzers, order of magnitude longer folded ion paths relax restrictions on detector response time, allowing implementation of new technologies that greatly improve dynamic range, detector lifetime, and ion detection efficiency. A detection system is presented, integrated into the Astral analyzer, that combines 10 keV post-acceleration and focal plane correction with a unique BxE focusing, optically coupled detector, pre-amplification and dual channel digitization. Calibration and peak handling methods are also described. The instrument demonstrated >1x104 dynamic range in a single shot, >100k resolving power, and a relative immunity to detector ageing.

Multi-reflection time-of-flight (MR-ToF) analyzers must control the transversal ion dispersion, orthogonal to the axis of reflection, conventionally via periodic refocusing, or more recently the shaped electrode structure adopted by the Astral analyzer. In principle, the complexity of the dispersion control on every oscillation may be avoided at a cost of a smaller number of oscillations, during which the dispersion doesn't exceed the limit of unrecoverable overlap. A method of dispersion control has been demonstrated experimentally and in simulation, whereby the ion beam is configured by a long focus trans-axial lens made of a pair of quasi-elliptical plates mounted above and below the ion beam, in order to optimize the spatial spread of the ions at a distant detector. The collimation concept was shown to effectively control beam expansion. For experimental confirmation, a prototype Astral ana-lyzer was modified, and resolving power above 70k demonstrated.

Multi-reflection time-of-flight (MR ToF) mass spectrometers are promising tools for accurate mass analysis, which combine a high resolving power with a short duty cycle. Though MR ToFs have been used for long in nuclear physics to explore short-lived isotopes, a restricted mass range of closed ion traps did not allow their use in chemical and bio-chemical applications, in which an analyte mixture contains a wide range of ionic species. This paper describes development of an open-path multi-reflection analyzer in which ion trajectories stay separated in the phase space on the oscillations to achieve an unlimited mass range. The design employs a controlled adiabatic ion drift along electrostatic mirrors. Ion focusing and time-of-flight aberration are corrected via reflection in slightly converging mirrors and refraction on optimally shaped electrostatic prisms.

Thermo Scientific™ Orbitrap™ analyzers represent prominent class of high-resolution mass analyzer commonly used in life sciences, and for interrogation of complex samples. Injected ions, trapped within a quadro-logarithmic field, orbit a central electrode and oscillate up and down the axis. A new class of multi-reflection time-of-flight mass analyzer has been developed based on the Orbitrap field structure plus an additional series of periodic lenses wrapped around the central axis to constrain beam dispersion. The axial and angular velocity of the injected ions was balanced so that with each axial oscillation, the ions passed through the next lens in the series, to form a tightly folded 25-metre long, 3-dimensional ion path, ending with ions striking a detector surface. Performance was interrogated via experiment and simulation. 70k resolving power was observed within the relatively compact analyzer, albeit at cost to transmission. A larger design with an integrated extraction trap and greater flight energy is discussed.

Reflectron-based time-of-flight analyzers rely on subnanosecond detector time response to achieve acceptable resolving power for low-mid-mass, multiple-ion peaks. With the adoption of multireflection analyzers, order of magnitude longer folded ion paths relax restrictions on detector response time, allowing implementation of new technologies that greatly improve dynamic range, detector lifetime, and ion detection efficiency. A detection system is presented, integrated into a multireflection analyzer, that combines 10 keV postacceleration and focal plane correction with a unique BxE focusing, optically coupled detector, preamplification, and dual-channel digitization. Calibration and peak-handling methods are also described. The instrument demonstrated >1 × 104 dynamic range in a single shot, > 100k resolving power, and a relative immunity to detector aging.

Space charge effects are the Achilles’ heel of all high-resolution ion optical devices. In time-of-flight mass analyzers these may manifest as reduction of resolving power, mass measurement shift, peak coalescence and/or transmission losses, whilst highly sensitive modern ion sources and injection devices ensure that such limits are easily exceeded. Space charge effects have been investigated, by experiment and simulation study, for the Astral multi-reflection analyzer, incorporating ion focusing via a pair of converging ion mirrors, and fed by a pulsed extraction ion trap. Major factors were identified as the resonant effect between ~103 ions of similar m/z in-flight, and the expansion of trapped packets of ~104-5 ions prior to extraction. Optimum operation and compensated ion mirror calibration strategies were then generated and described based on these findings.

Optimizing data-independent acquisition (DIA) methods for proteomics applications often requires balancing spectral resolution and acquisition speed. Here we describe a real-time, full mass range implementation of the Phase-constrained Spectrum Deconvolution Method (ΦSDM) for OrbitrapTM mass spectrometry that increases mass resolving power without increasing scan time. Comparing its performance to the standard enhanced Fourier transformation (eFT) signal processing revealed that the increased resolving power of ΦSDM is beneficial in areas of high peptide density and comes with a greater ability to resolve low-abundance signals. In a standard 2-hour analysis of a 200 ng HeLa digest, this resulted in an increase of 16% in the number of quantified peptides. As the acquisition speed becomes even more important when using fast chromatographic gradients, we further applied ΦSDM methods to a range of shorter gradient lengths (21, 12, and 5 min). While ΦSDM improved identification rates and spectral quality in all tested gradients, it proved particularly advantageous for the 5 min gradient. Here the number of identified protein groups and peptides increased by >15% in comparison to eFT processing. In conclusion, ΦSDM is an alternative signal processing algorithm for processing Orbitrap data that can improve spectral quality and benefit quantitative accuracy in typical proteomics experiments, especially when using short gradients.

Multi-pass zoom-mode methods have been described to extend the flight path and resolving power of multi-reflection time-of-flight mass spectrometers, by deflecting exiting ions back into repeat passes through the analyzer. Such methods have been adapted to the Astral analyzer, with repeated passes through a prism deflector found to compensate resolution-destroying aberrations. Changes to resolution were simulated and experimentally characterized, with more than a doubling of resolving power observed with only a moderate loss of transmission. Further methods are also proposed to eliminate the mass spectrum uncertainty caused by overlapping of signals from ions that have undergone unknown numbers of passes.

The quality of data in charge detection mass spectrometry depends on accurate determination of ion charge. While the method of selective temporal overview of resonant ions (STORI) has proven to be highly enabling for determining the charge of ions that survive for variable amounts of time, it assumes that the ion frequency exactly matches the frequency being used in the calculation. Any mismatches result in low charge estimates. To address this, the misSTORI method was developed to correct these discrepancies. This can significantly reduce the charge measurement errors for samples with unstable masses. As an example, the misSTORI approach can eliminate a 5.7% charge determination error for a VP3-only AAV capsid that shifts 25 ppm in mass.

The measurement of collision cross sections (CCS) offers supplemental information about sizes and conformations of ions beyond mass analysis alone. We have previously shown that CCSs can be determined directly from the time-domain transient decay of ions in an Orbitrap mass analyzer as ions oscillate around the central electrode and collide with neutral gas, thus removing them from the ion packet. Herein, we develop the soft sphere collision model, thus deviating from prior FT-MS CCS hard sphere model, to determine CCSs as a function of center-of-mass collision energy in the Orbitrap analyzer. With this model, we aim to increase the upper mass limit of CCS measurement for native-like proteins, characterized by low charge states and presumed to be in more compact conformations. We also combine CCS measurements with collision inducing unfolding and MS/MS experiments to monitor protein unfolding and disassembly of protein complexes and measure CCSs of ejected monomers from protein complexes.

The growing trend toward high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass-resolving quadrupole, the Orbitrap, and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared with state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with a high dynamic range and resolution, synchronized with the Astral analyzer's acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers.

Ion traps are routinely directly coupled to mass analyzers, where they serve to suitably cool and shape an ion population prior to pulsed extraction into the analyzer proper. Such devices benefit from high duty cycle and transmission but suffer slow ion processing times caused by a compromise in the buffer gas pressure range that suitably dampens the ion kinetic energy without causing excessive scatter during extraction or within the analyzer. A rectilinear RF quadrupole ion trap has been characterized, conjoining a pressurized collision region with a pumped extraction region, and an unbroken RF interface for seamless ion transfer between them. Auxiliary electrodes mounted between the RF electrodes provide DC voltage gradients that serve to both guide ions through the device and position them at the extraction slot. The influence of the auxiliary DC upon the trapping RF field was measured, and suitable parameters were defined. A mode of operation was developed that allowed parallel processing of ions in both regions, enabling a repetition rate of 200 Hz when the device was coupled to a high-resolution accurate-mass analyzer.

Mass spectrometry (MS)-based thermal stability assays have recently emerged as one of the most promising solutions for the identification of protein-ligand interactions. Here, we have investigated eight combinations of several recently introduced MS-based advancements, including the Phase-Constrained Spectral Deconvolution Method, Field Asymmetric Ion Mobility Spectrometry, and the implementation of a carrier sample as improved MS-based acquisition approaches for thermal stability assays (iMAATSA). We used intact Jurkat cells treated with a commercially available MEK inhibitor, followed by heat treatment, to prepare a set of unfractionated isobarically-labeled proof-of-concept samples to compare the performance of eight different iMAATSAs. Finally, the best-performing iMAATSA was compared to a conventional approach and evaluated in a fractionation experiment. Improvements of up to 82% and 86% were demonstrated in protein identifications and high-quality melting curves, respectively, over the conventional approach in the proof-of-concept study, while an approximately 12% improvement in melting curve comparisons was achieved in the fractionation experiment.

Measurement of collision cross section (CCS), a parameter reflecting an ion's size and shape, alongside high-resolution mass analysis extends the depth of molecular analysis by providing structural information beyond molecular mass alone. Although these measurements are most commonly undertaken using a dedicated ion mobility cell coupled to a mass spectrometer, alternative methods have emerged to extract CCSs directly by analysis of the decay rates of either time-domain transient signals or the FWHM of frequency domain peaks in FT mass analyzers. This information is also accessible from FTMS mass spectra obtained in commonly used workflows directly without the explicit access to transient or complex Fourier spectra. Previously, these experiments required isolation of individual charge states of ions prior to CCS analysis, limiting throughput. Here we advance Orbitrap CCS measurements to more users and applications by determining CCSs from commonly available mass spectra files as well as estimating CCS for multiple charge states simultaneously and showcase these methods by the measurement of CCSs of fragment ions produced from collisional activation of proteins.

A quasi-analytical approach to solving the system of transport equation describing the evolution of charged particle ensembles in neutral gas flow under the action of external electric fields is developed, with allowance for Coulomb interaction and recombination effect. First, a closed solution for arbitrary initial distributions of ions is obtained for the case of constant ion velocities. Then, using a simple numerical procedure, this result is generalized to the case of variable velocities as applied to arbitrary distributions of external electric fields and gas flow velocities. Finally, a self-consistent problem for a system of transport equations, which, in addition to the recombination effect, include the Coulomb interaction between ions, is considered, based on the method of characteristics. The original system of partial differential systems is reduced to a system of ordinary differential equations along the characteristics, exact solutions for some partial cases are obtained, and peculiarities of the use of the perturbation method are discussed.

The rapidly increasing adoption of high-resolution accurate-mass methods in analytical laboratories has fueled demand for instruments that combine high performance and reliability with small size and greater ease-of-use. This paper presents the major design principles that are driving the evolution of the hybrid quadrupole-Orbitrap instrument architecture to enable a greater range of applications and users. These principles may be summarized as follows: better usage of physical space and better access for service by means of size reduction of pumping and ion optics; expanded use of technologies from electronics in ion-optical design; flexibility in performance via modularity of design of the hardware and software components; and, harmonization of interfaces with other instruments to facilitate sharing and transferability of analytical workflows. The design of a novel family of hybrid mass spectrometers is described in detail, and performance evaluation is carried out on a wide variety of samples for its three representatives: the Orbitrap Exploris 120, Orbitrap Exploris 240 and Orbitrap Exploris 480 mass spectrometers.The new instrument family is shown to offer compelling potential not only for high-end proteomics and biopharmaceutical applications, but also for screening, trace, targeted and clinical analysis by liquid chromatography/mass spectrometry methods.

The paper deals with space-charge interactions in the ion population trapped in the Orbitrap[Formula: see text] mass analyzer where the ions perform multiple quasi-harmonic oscillations in the axial direction. The many-particle problem for interacting ions is mathematically complicated and its solution, even numerical, is obstructed by the required precision of one per million to be maintained on a large number [Formula: see text] of oscillation periods. We develop a perturbation method based on the Bogoliubov–Krylov–Mitropolsky theory and derive averaged Hamiltonian equations in perturbations, which describe the evolution of the ions’ oscillation amplitudes and phases in so-called “slow” time. This approach provides a semi-analytical comprehensive model of resonant and nonresonant space-charge effects and allows fast and accurate numerical computation. Practical mitigation strategies for most deteriorating space-charge effects like coalescence and frequency shifts are considered.

This chapter discusses fundamentals of the Orbitrap™ mass spectrometry—the use of a purely electrostatic ion trap with harmonic properties to store ions and analyze their mass-to-charge distributions. The first section demonstrates principles of ion confinement in the quadro-logarithmic electrostatic field, where the ions perform hundreds of thousands of oscillations without losing their common phase. The discussion covers practical aspects of ion injection into the trap and the image current detection. The subsequent section introduces the reader to the Orbitrap aberration theory, gives a classification of electric field perturbations, and outlines the compensation methods. The next section concerns signal processing methods of the detected transients and covers both the traditional Fourier transform approaches and super-FT resolving methods. The chapter concludes with the overview of historical milestones of the Orbitrap technology and selected applications.