The Proteomics Core Facility provides protein characterization by mass spectrometry. This includes identification of unknown proteins, quantitative comparison of proteins in biological samples, and mapping of post-translational protein modifications. Instrumentation associated with the Proteomics Core includes the following.
Orbitrap Tribrid Mass Spectrometers
The Thermo Orbitrap Eclipse Tribrid system (Thermo Scientific) is the latest generation of the state-of-the-art Orbitrap mass spectrometry technology. The facility operates two Eclipse systems as well as two Orbitrap Fusion Tribrid instruments. The Orbitrap Tribrid systems combine a quadrupole mass filter with Orbitrap and linear ion trap mass analyzers in a novel configuration that maximizes ion transfer efficiency, allowing flexible combinations of different fragmentation techniques, scanning modes, and mass analyzers. The quadrupole mass filter allows precursor ion selection, enabling parallel use of the mass analyzers and synchronous precursor selection for MS3 analysis. An ion-routing multipole serves as a higher-energy collisional dissociation (HCD) cell and also transfers ions efficiently between the Orbitrap and linear ion trap mass analyzers, allowing use of either mass analyzer in combination with any ion fragmentation technique. The ultra-high-field Orbitrap mass analyzer has a resolving power of up to 500,000 FWHM and 45 Hz scan rate. A linear ion trap performs collision-induced dissociation (CID) and electron-transfer dissociation (ETD).
Orbitrap Exploris 480 Mass Spectrometer
The Orbitrap Exploris 480 mass spectrometer (Thermo Scientific) combines a quadrupole mass filter with a high-field Orbitrap mass analyzer and high-capacity ion transfer capability to optimize scan rate, sensitivity, and resolution. Improvements over earlier generation instruments in this class include significantly enhanced instrument sensitivity largely due to improved ion transfer capabilities, improved instrument robustness and serviceability, and compatibility with next-generation ion sources such as the FAIMS Pro (high field asymmetric ion mobility spectrometry) ion source. The facility is equipped with two of these instruments, which are used primarily for data-independent acquisition (DIA).
TSQ Quantiva Mass Spectrometer
The TSQ Quantiva (Thermo Scientific) is a triple quadrupole instrument used exclusively for selected reaction monitoring (SRM) experiments for targeted quantitative proteomics. The advantage for SRM experiments on the TSQ Quantiva is rapid switching between transitions with a set dwell time. The result is that a large number of peptides can be measured in a single run with a consistent 1.5 cycle per second acquisition rate, which gives at least 20 data points for all chromatographic peaks. Scheduling is used for all experiments based on the known retention times of targeted peptides. SRM on a triple quadrupole mass spectrometer remains a workhorse experiment for quantitative analysis with outstanding precision, lower limits of detection, and robust instrument operation.
Q Exactive Plus Mass Spectrometer
The Q Exactive Plus (Thermo Scientific) is a hybrid quadrupole-orbitrap instrument used for parallel reaction monitoring (PRM) in our targeted quantitative proteomics services. Orbitrap instruments are known for high m/z resolution, with values ranging from 17,500 to 280,000 on the Q Exactive Plus. The advantage for PRM experiments on the Q Exactive Plus 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.
UltiMate 3000 Binary Analytical Liquid Chromatography System
The UltiMate 3000 UHPLC system (Thermo Scientific) is capable of precise and efficient offline fractionation of complex protein digests. This system is used in the facility workflow to fractionate peptides at basic pH in order to complement the acidic pH separation performed during liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. The UltiMate 3000 can sustain pressures as high as 9000 psi and flow rates as low as 50 nL/min. The system includes an analytical autosampler and fraction collector to allow efficient processing of large numbers of samples, as well as a UV detector to monitor peptide elution and prevent cross-contamination.