A Novel Family of Quadrupole-Orbitrap Mass Spectrometers for a Broad Range of Analytical Applications

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.


INTRODUCTION
Mass spectrometry has long established itself as a universal analytical technique for a breathtaking range of applications, spanning from trace elemental, environmental, food, anti-doping and forensic analysis, to analysis of proteins, biopharmaceuticals and even intact viruses. While benefiting greatly from innovations in separation, ionization and data processing technologies, mass spectrometry instrumentation has also undergone multiple fundamental changes in mass analyzer technology and instrument architectures that enabled it to repeatedly expand its analytical power and application reach. One of the key trends of the last two decades is the ever-accelerating transition to highresolution accurate-mass (HRAM) analysis.

EXPERIMENTAL SECTION.
All data was generated using Thermo Scientific™ Orbitrap Exploris™ 120, Orbitrap Exploris 240, or Orbitrap Exploris 480 mass spectrometers (Thermo Fisher Scientific, Bremen, Germany), as indicated below. Unless specified otherwise, performance is similar across all models.
Initial calibration of all instruments was performed using Thermo Scientific™ Pierce TM FlexMix TM calibration solution, a mixture of 16 highly pure, ionizable components (mass ranges: 40 to 3000 m/z) developed for both positive and negative ionization calibration.
For pesticide samples, Thermo Scientific™ HyperSep™ Dispersive Solid Phase Extraction clean-up products were used for a simplified QuEChERS extraction protocol, with 1 µL of sample injected at 300 µL/min flow rate using a Thermo Scientific™ Vanquish Flex Binary ultra-high-performance liquid chromatograph (UHPLC) with Accucore™ aQ C18 column (2.1mm ID × 100mm, 2.6 µm particles) sustained at 30°C. Thermo Scientific™ TraceFinder™ 5.1 software and mzCloud Offline Spectral Library for mzVault 2.3 (Pest_Herb_2020A) was used for processing of targeted analysis data and Thermo Scientific™ Compound Discoverer™ 3.1-for processing of untargeted data.
Metabolomic analysis was carried out for NIST SRM1950 human plasma standard with 15 min gradient elution using a Thermo Scientific™ Hypersil GOLD™ column (2.1 mm ID ×150 mm, 1.9µm particle size) at a flow rate of 300 µL/min in line with a Thermo Scientific™ Vanquish™ Horizon UHPLC system. Data were processed using Compound Discoverer software for unbiased peak detection and identification.
Benchmarking experiments for trastuzumab (Herceptin®) monoclonal antibody were carried out using the Vanquish Horizon UHPLC with a MabPac SEC-1 (4.0 mm ID ×150 mm) column for native analysis and a MAbPac RP (2.1 mm ID × 100 mm, 4 µm particles) column for intact and sub-unit analysis.
Digestion to subunits was performed using FabRICATOR® (IdeS) enzyme (Genovis AB, Lund, Sweden). Data were processed with Thermo Scientific™ BioPharma Finder™ 4.0 software using ReSpect and Xtract intact mass deconvolution algorithms (for isotopically unresolved and resolved spectra, respectively).
Proteomic benchmarking experiments were carried out on Thermo Scientific™ Pierce TM HeLa Protein Digest Standard using the Thermo Scientific™ EASY-nLC™ 1200 UHPLC system with Thermo Scientific™ EASY-Spray™ source and ES803A column. Alternatively, the Ionopticks Aurora series UHPLC Column (75 µm ID × 250 mm) with the Thermo Scientific™ Nanospray Flex™ ion source was employed. Spectral library search was carried out using Thermo Scientific™ Proteome Discoverer™ 2.4 software for data-dependent acquisition experiments and Spectronaut 14 software (Biognosys, Zurich, Switzerland) for data-independent acquisition experiments. In all experiments a false discovery rate of 1% was applied for both peptides and proteins. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 June 2020 doi:10.20944/preprints202006.0111.v1

Main Principles of the Instrument Architecture
The new family of mass spectrometers was designed following several general principles. The first principle is the modularity of the design, wherein each module underwent -somewhat paradoxicallydeeper integration into a self-contained unit.
The instrument presents a user with an LC-compatible ion source connected to an atmosphere-tovacuum interface. In this work, two variants of this module are discussed in detail: the first includes a round-bore metal transfer tube followed by an S-lens, while the second includes a high-capacity transfer tube (HCTT) with an electrodynamic ion funnel. Each interface is fully compatible with any of the ion sources from the Tribrid and TSQ triple quadrupole mass spectrometers. This commonality with the latest Tribrid and TSQ instruments was another major design principle of the new family and can be observed in many aspects of its operation. On the source side, this allows interfacing to the same Tribrid front-end options, such as High-Field Asymmetric Waveform Ion Mobility Spectrometry (embodied in the FAIMS Pro™ interface) or the EASY-IC™ internal lock-mass ion source.
Following the source module, the analyzer super-module forms the heart of the instrument. It contains several modules: an S-shaped bent flatapole, a hyperbolic-rod segmented quadrupole mass filter, an Independent Charge Detector (ICD) module, a C-trap/Orbitrap block with an ultra-high field Orbitrap mass analyzer and finally an Ion-Routing Multipole (IRM) module. All modules are aligned along a common axis and are easily accessible from the top of the chamber ( Figure 1). One of the striking features of this super-module is the extensive use of printed-circuit boards (PCBs) as vacuum feedthroughs with predominantly spring-loaded connections, and also as elements of ion optics.
Usage of PCBs results in the elimination of tens of cables, -which in turn removes one of the major reasons for electrical failures.
The compact design of the analyzer super-module is directly enabled by one of the most important technical breakthroughs realized in this development: the turbomolecular pump (TMP) module. In all previous families of Orbitrap instruments, the built-in requirement of ultra-high vacuum (UHV) in the 10 -10 or even 10 -11 mbar range for Orbitrap analysis has necessitated a bulky pumping system with several pumps, and this imposed the major constraint on overall design and size of the instrument. In the new instrument, a single, purpose-developed, six-stage TMP evacuates the entire analyzer supermodule. This design results in a drastic reduction of instrument footprint and volume, while alignment of all ion-optical components along a common axis allows for easier access from the sides for electrical connections and from above for servicing. Overall, ease of access and service were primary considerations at every step of the design process.
The analyzer resides within a sheet-metal frame and is contacted from all sides by easy-to-remove electronic boards with spring-loaded contacts. These vertical boards are plugged via PCIe connectors into a horizontal base-plane board at the bottom of the instrument, which provides the connections to power as well as internal and external communication. This electronic module embodies the next 6 design principle: extensive on-board intelligence combined with both remote and internal diagnostic capabilities.
It should be noted that both analyzer and frame allow access from the rear of the mass spectrometer for any potential extensions of the instrument through the IRM in the future.
Detailed design and operation description for this novel family of high-performance, fit-for-purpose quadrupole-Orbitrap instruments is presented in Section 1 of Supplemental Information. This includes the Orbitrap Exploris 120 MS, optimized for quantitative environmental, food safety, and toxicology analysis, as well as other small-molecule applications, and the universal Orbitrap Exploris 240 and 480 MS systems, delivering enhanced performance also for -omics, pharmaceutical and biopharmaceutical applications. Performance characterization of all Orbitrap Exploris models was conducted for their most relevant applications, with selected results presented below.

Stability of Mass Accuracy
One of the most important characteristics of the Orbitrap mass analyzer since its inception is its high mass accuracy and mass stability. This figure-of-merit is confirmed as shown in Figure 2 for continuously repeated analysis of VetDrug samples using the Orbitrap Exploris 120 instrument, with a subset of 16 substances in a mass range m/z 160 to 900 evaluated for mass accuracy. While mass stability following system calibration is specified for 24 hours, in standard air-conditioned laboratories measured masses usually remain accurate for much longer, e.g., for several days as shown in Figure   2a.
Full scan MS analysis with scan-by-scan internal calibration, using the integrated EASY-IC source to produce the lock-mass ion, was performed to assess the achievable mass accuracy and duration of

Scan-to-scan Polarity Switching
The acquisition scan rate with scan-to-scan polarity switching was accelerated, relative to the Q Exactive instruments, by reducing settling times of ion optics. Shown in Figure 4, a full MS (30,000 resolution) and targeted MS/MS (15,000 resolution) in each positive and negative mode are acquired.
The dual polarity MS and MS/MS cycle took an average of less than 0.6 seconds (Fig. 4a). The precursor mass error stayed in low ppm for both positive and negative modes even with external mass calibration, demonstrating that high mass accuracy is retained during polarity switching ( Fig. 4b and 4c). The fragment ions in the targeted MS/MS also demonstrated low-ppm mass accuracy (data not shown).
Quantitative Performance on the Example of Pesticide Analysis Initial results for tandem mass tag quantitation in short gradients were presented in [12]. As the DIA paradigm evolved over the last years to deliver increased accuracy, quantitative precision as well as proteome coverage with improved throughput, its requirements for higher ion loads in broader isolation windows appeared to be really well suited to the high space charge capacity and increased speed of the C-trap/Orbitrap combination, as illustrated in [12] for very short (<30 min) gradients with the Orbitrap Exploris 480 MS.
One of the general hurdles to wider adoption of DIA is the need for cumbersome project-specific library creation. Recently, promising new approaches have been proposed and tested to overcome this limitation utilizing deep neural networks in DIA library creation. The library created in-silico by, e.g., the PROSIT engine is adjusted with the help of a limited number of gas-phase fractionated DIA runs as described in [14][15]. As the result, the Orbitrap Exploris 240 instrument, even without the          The return transfer of ions from the IRM into the C-trap was optimized for high ion loads and throughput. Specifically, a pre-purge pulse is applied, lowering the DC offset of the IRM rods to move away any ions residing close to the IRM entrance lens, so that they will experience a proper focusing field during the ensuing purge event. Once rapid elevation of the IRM offset and axial field initiates ion transfer, the IRM offset, the axial field, and the C-trap lens voltages continue to be slowly ramped up during the transfer so that the ions are subjected to an increasing potential barrier as they approach lenses during reflections within the C-trap and therefore cannot get deposited onto lens edges. The resulting digital waveform is then processed by a built-in PC using an enhanced version of Fourier Transformation (eFT™) as described in [6]. For Tandem Mass Tag™ (TMT™) experiments, narrow m/z regions near TMT-10 reporter peaks [7,8] are processed by the Phase-constrained signal deconvolution method (Fi-Transform) as described in [9]. instruments contain also provisions for remote system monitoring by the service organization as well as by users using Thermo Scientific™ Almanac web-based application.
This work presents three different high-resolution accurate mass LC/MS instrument models based on the new design that collectively address a broad range of important analytical applications.
The Orbitrap Exploris 120 MS is designed for highest performance in quantitative environmental, food safety and toxicology analysis, as well as other small-molecule applications. It provides resolving powers from 15,000 to 120,000 at m/z 200 (32 to 256 ms transients, respectively) and has a white panel on the front instead of a black one of Figure 1a. It utilizes a round-bore transfer tube with 580 µm ID followed by an S-lens operated at 650 kHz. Its pumping system consists of a six-stage TurboVac HexaInlet TMP and a single-stage rotary vane pump Sogevac SV65BI (both from Leybold GmbH, Cologne, Germany). Its IRM pressure is fixed at a standard setting of 1.1 . 10 -2 mbar.
The Orbitrap Exploris 240 MS is designed as a universal instrument that could also address -omics, pharmaceutical and bio-pharmaceutical applications. It provides resolving powers from 15,000 to 240,000 at m/z 200 (32 to 512 ms transients, respectively) and features a silver front panel on Figure   1a. It also utilizes a round-bore transfer tube with 580 µm ID followed by an S-lens operated at 650 kHz. asymmetric waveform ion mobility spectrometer (FAIMS). Details of the design and characterization of the FAIMS Pro interface are presented in [11]. A unique feature of this device is the use of cylindrical electrodes that help focus ions through the electrode assembly as they are transported by the nitrogen carrier gas from inlet to outlet. With the inner electrode blocking the "line-of-sight" for clusters and dust particles, the interface provides an additional benefit of protection from environmental factors.
As shown in [10],    undergoing full cleaning and update of software between study 1 and 2. Instrument 2 (3rd study) ran in parallel to 2nd study to confirm robustness results. The red stars indicate cases when performance decline was rectified by maintenance of UHPLC or simply transfer tube cleaning.
Further experimental details are presented in Section 2.      glycoforms of the heavy chain (b) were obtained with low-ppm mass accuracy following deconvolution of the corresponding full scan spectra of the light (c) and the heavy chain (e), with chromatogram shown in (d). A high-resolution setting of 240,000 was used for the light chain and then switched to a low resolution of 15,000 during heavy chain elution. Isotopic resolution of the heavy chain was achieved in selected ion monitoring mode at 480,000 resolution setting (f).
Subsequent top-down analysis using HCD fragmentation in the IRM at different collision energies resulted in cleavage of 62% residues in the light chain and 33% residues in the heavy chain. In each experiment, 200 ng Pierce HeLa digest was analyzed with a 120 min LC gradient using a Top20 DDA method and all other experimental conditions identical to those in Figure 9. In each experiment 1000 ng Pierce HeLa digest was analyzed with a 60 min LC gradient using a Top20 DDA method and EASY-Spray ES803A column.