One Orbitrap Astral LC-MS System
The Orbitrap Astral mass spectrometer features significant advancements over previous generation Orbitrap instruments in sensitivity and acquisition speed. The key novel feature of this instrument is the Astral mass analyzer, with a 30-meter asymmetric time-of-flight path capable of resolution up to 80,000 FWHM downstream of a 200 Hz ion processor and an ion foil that tightly focuses the ion beam with minimal transmission loss. In combination with an ultra-high-field Orbitrap mass analyzer, the hybrid instrument can achieve greater depth with less sample in shorter acquisition times than previously possible. The Orbitrap Astral will be used primarily for data independent acquisition (DIA) quantitative proteomics. The Orbitrap Astral instrument is equipped with the following accessories:
- Vanquish Neo UHPLC System: Reliable, high-resolution peptide chromatography at submicroliter per minute flow rates is required to achieve optimum performance with the Orbitrap Astral mass spectrometer. The UHPLC system is equipped with an autosampler to allow highthroughput operation, and a binary solvent manager to allow gradient separation of peptides during data acquisition.
- Nanospray Ion Source: The nanospray ion source allows efficient electrospray ionization of peptides at UPLC flow rates in the 200-300 nl/min range in conjunction with the UltiMate 3000 RSLCnano and nanoAcquity UPLC systems. This capability is critical to maximizing proteomic depth under sample-limiting conditions. In addition, mapping of PTMs, biomarker discovery, and interaction proteomics research are not feasible without these low flow rates.
- EASY-IC (internal calibration) Ion Source: This option increases mass accuracy using internally generated ions for calibration. Optimum mass accuracy is critical to confident assignment of ions in all mass spectrometric data.
Two Orbitrap Fusion LC-MS Systems
The Orbitrap Fusion mass spectrometer instrument design combines a quadrupole mass filter with Orbitrap and linear ion trap mass analyzers in a novel configuration that maximizes ion transfer efficiency, allowing flexible combination of different fragmentation techniques, scanning modes, and mass analyzers. Collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron-transfer dissociation (ETD) can be applied at any stage of MSn analysis using either mass analyzer. Parallelization and synchronous precursor selection capabilities increase the quality of data that can be acquired, and improved mass analyzer and detector designs increase the sensitivity and dynamic range of the instrument compared with earlier-generation Orbitrap systems and other options currently on the market. The Fusions are used mostly for data dependent acquisition (DDA) quantitative proteomics. All Orbitrap Fusion-class mass spectrometers maintained and operated at UAMS are equipped with a nanospray ion source and EASY-IC (both as detailed above) plus the following options and accessories:
- UPLC System Interface: Each Orbitrap Fusion-class mass spectrometer at UAMS is integrated with an UltiMate 3000 RSLCnano system (Thermo). Each UPLC is equipped with an autosampler to allow high-throughput operation, and a binary solvent manager to allow gradient separation of peptides during data acquisition. Each system is configured with a 0.5 cm trapping column and a 15 cm analytical column. New Objective IntegraFrit (trapping) and PicoFrit (analytical) columns (75 um internal diameter) are packed on-site with reverse phase XSelect CSH C18 2.5 um resin.
- Electron-Transfer Dissociation (ETD) Source: ETD is essential for comprehensive identification and mapping of PTMs due to the unbiased nature of ETD peptide backbone fragmentation and the relative stability of modifications on peptides fragmented by ETD as compared to CID and HCD.
Two Orbitrap Eclipse LC-MS Systems
The Orbitrap Eclipse Tribrid mass spectrometer (Thermo Scientific) has a redesigned segmented quadrupole mass filter with improved precursor sensitivity and selectivity compared to the earlier generation Orbitrap Fusion, high mass range MSn capability and proton transfer charge reduction to improve analysis of intact proteins and protein complexes, and real-time database search capability to improve depth and accuracy of tandem mass tag (TMT) analysis. As with the Fusion systems described above, different collision and scanning modes can be utilized in flexible combinations to optimize instrument performance for specific sample types and workflow. The Eclipse systems in our facility are used primarily for TMT quantitative proteomics, with MultiNotch synchronous precursor selection used to generate reported ion data at the MS3 level from multiple HCD fragment ions at the MS2 level. All Orbitrap Eclipse LC-MS systems are equipped with nanospray ion sources, EASY-IC, and UltiMate 3000 LC systems as detailed above for the Fusion systems. One of the Eclipse systems is also equipped with ETD ionization capability.
Two Orbitrap Exploris 480 LC-MS Systems
The Orbitrap Exploris 480 mass spectrometer features the high-capacity ion transfer tube and electrodynamic ion funnel found in the Orbitrap Tribrid instrument systems, along with a shorter quadrupole design to optimize ion transmission efficiency. The Exploris 480 system also features BoxCar acquisition capability ideally suited for implementation of data independent acquisition (DIA) workflows. The system is also capable of target selected ion monitoring and multiplexing. The Exploris instruments in our facility are used primarily for DIA quantitative proteomics. These instruments are equipped with nanospray ion sources, EASY-IC, and UltiMate 3000 LC systems as detailed above for the Fusion systems.
Two UltiMate 3000 UHPLC Systems
The facility maintains and operates two stand-alone UltiMate 3000 UHPLC systems (Thermo Scientific) for offline fractionation of samples at the peptide level prior to LC-MS/MS analysis. The systems allow reliable, high-quality, high-throughput peptide separations in order to efficiently decrease the complexity of each LC-MS/MS run within a single or multiplexed sample analysis. Each UltiMate 3000 system is equipped with an autosampler and fraction collector to allow high-throughput processing, a column heater, and UV detector to allow real-time monitoring of peptide separation. Complex peptide samples are separated under basic pH conditions on a 100 x 1.0 mm Acquity BEH C18 column (Waters). Combined with the in-line separation described above (UPLC system interface), performed under acidic pH conditions, this makes possible an efficient two-dimensional peptide separation compatible with high-throughput sample analysis and efficient use of instrument time.
Sample Preparation Equipment
Tecan Freedom EVO 150
This system is compatible with a broad range of sample preparation workflows commonly used in proteomics and is thus ideal for a rapidly expanding facility and compatible with the type of continuous workflow development necessary to maintain cutting-edge capability. The system offers a precision liquid handler with positive-displacement probe syringes, a vacuum separation chamber, a heated orbital shaker, a 96-well magnetic separating module, and a software package including pre-optimized sample preparation protocols. The system allows the reformatting of samples from tubes to 96 well plates for increased efficiency. These features will allow our facility to further automate and scale existing sample processing and cleanup protocols and further minimize inefficiencies associated with transitions between protocol steps. Compatibility with plate-based autosamplers and Evosep Evotip Pure tips will also reduce the amount of time required to prepare processed samples for data acquisition on our existing LC-MS instrument systems.
Evosep One Liquid Chromatography System
This system features a disposable trap column that allows offline desalting of samples prior to elution of the sample from the same column by the Evosep autosampler. This capability reduces the amount of time spent on manual sample cleanup and integrates two previously separate processes into a single automated cycle. Given the increasing utilization of data-independent acquisition (DIA) analysis, the ability to connect the Evosep system with the Orbitrap Astral or Exploris 480 mass spectrometer gives us increased flexibility to adapt the throughput capability of the DIA platform in our facility to meet demand.
Tecan Resolvex A200
This system is a stand-alone, gas-based positive pressure processor which allows rapid and reliable digestion or desalting of protein or peptide samples in 96-well plate format. The Resolvex system is particularly useful for resin-based protocols requiring consistent solvent delivery to ensure reproducibility.
Opentrons OT-2
This system provides flexible, fully configurable, plate-compatible automation with single and eight-channel pipetting options. The open-source programming interface allows for straightforward modification of standard protocols for unusual sample sets or new protocol optimization.
Uninterruptible Power Supply
All mass spectrometers and associated LC and computer systems are equipped with AMETEK Powervar Security Plus uninterruptible power supplies to ensure continuous operation in the event of building power failure and to protect the instrument systems from power surges or fluctuations.
