One Orbitrap Exploris 480 LC-MS System
OMRF is in the process of acquiring an Orbitrap Exploris 480 hybrid quadrupole-orbitrap mass spectrometer during Year 5 of the current R24 funding cycle. Space, air conditioning, power, and ventilation are already in place. The Exploris will be used as a replacement for the parallel reaction monitoring (PRM) experiments on the Q Exactive Plus instrument described below. The instrument will be equipped with a FAIMS Pro Duo interface and a Vanquish Neo UHPLC.
One Stellar LC-MS System
A new Thermo Scientific Stellar tandem mass spectrometry system is used for parallel reaction monitoring (PRM) experiments for targeted quantitative proteomics. The advantages of PRM experiments on the Stellar are related to unique features of the quadrupole-linear ion trap system that provide high sensitivity and extraordinarily rapid data acquisition rates – in the range of 100hz. The result is the capability to measure 5000 peptides and therefore 2500 proteins in each run. Importantly, new software tools streamline the conversion of DIA results from the Exploris 480 into PRM methods on the Stellar. Together these instruments keep the Targeted workflow at the state of the art for sensitivity and data acquisition rates needed for advanced, high throughput targeted proteomics experiments.
One Q Exactive Plus LC-MS System
The hybrid quadrupole-orbitrap instrument is used for PRM in our targeted quantitative proteomics services. The advantages of the PRM experiment is the high-resolution product ion m/z scan and the increased specificity of detection this m/z resolution provides via smaller m/z tolerances in the data analysis. The hybrid quadrupole-orbitrap instruments are also more sensitive than the TSQ Quantiva. As a result, the resource uses PRM for our lower abundance measurements, including all PTM measurements. It is equipped with an UltiMate 3000 LC.
Sample Preparation Equipment
The OMRF laboratory has all the equipment necessary to prepare and store protein samples for mass spectrometric analysis, including an Opentrons OT-2 automated liquid handling system, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) apparatus, power supplies, incubators, microcentrifuges, sonicators, speedvacs, and −20°C and −80°C freezers.
Uninterruptible Power Supply
Systems are powered by dedicated UPS systems, Toshiba 1600EP and Powervar Security Plus.
Local Data Analysis and Storage
Each instrument is equipped with a Dell workstation for instrument operation and initial data storage. The laboratory has Dell Optiplex workstations for all laboratory personnel with additional systems available by network for data processing. The laboratory uses Skyline (MacCoss Lab, UW Genome Sciences) for the basic data processing. All raw data and processed results are stored at multiple hardware, cloud, and network locations. The laboratory maintains a local copy of all data on simple external hard drives stored in the laboratory and using unlimited Dropbox space provided by OMRF. Data are also saved to network servers maintained and backed up by the OMRF IT group. The IT at OMRF provides secure and centralized server hosting for bioinformatics and high-performance computing. These systems are hosted within a local “cloud” environment. Four servers form a VMware vSphere cluster are the basis for this private “cloud”. In total, there is over 20TB of RAM on more than 2000 CPU cores. Primary storage is provided by a multi-node high performance network attached storage cluster from Dell EMC Isilon. The current primary Isilon cluster provides 500TiB of primary storage, with the ability for easy expansion by adding additional nodes to the storage cluster. A separate storage cluster using NVMe 130TiB of dedicated storage for scratch. A third 740TiB storage system is kept in a secure offsite location for backup of these systems. Data are written in triplicate to this system in distinct separate geographic regions to protect against catastrophic failure of systems and sites. All machines are protected by a combination of network edge firewall with a policy that denies traffic by default, computer system local software firewall, and network switch access control lists. Computers and software are kept current with upgrades, patches, and security releases as necessary.
