University of Pittsburgh Cancer Institute (UPCI)

Equipment

The Proteomics lab of the MSP occupies 2400 ft² on the 4th floor of the Magee-Womens Research Institute and is equipped with comprehensive laboratory resources and instrumentation for proteomics research. The key proteomics MSP resources are:

2 ThermoElectron LTQ XLs (one with electron transfer dissociation) linear ion trap mass spectrometers: A high performance linear ion trap (LIT) mass spectrometer (MS) that has coupled online a nanoflow HPLC that, together, provide for robust acquisition of tandem MS data. This LIT-MS represents a leap in performance. The segmented linear trap with radial ejection and dual detection system enables the highest overall detection efficiency resulting in unparalleled sensitivity. These capabilities combined with rapid scanning available through state-of-the-art electronics results in information rich data collection at breakthrough speed. Superior sensitivity and fast scanning produces better peak shape resulting in better quantitation over a wider linear dynamic range. The enhanced MS/MS affords excellent higher order MS spectra that rapidly facilitates both metabolic and proteomic structural elucidation

ThermoElectron TSQ ULTRA triple quadrupole mass spectrometer with FAIMS source: This instrument is coupled to a nanoflow HPLC allowing for automated quantification of small molecules and peptides alike. The instrument features the new Ion MAX source ESI and APCI probes, a titanium skimmer with superior skimming and larger aperture for increased robustness and sensitivity. Its ion transfer tube lens with wider aperture provides enhanced ion focusing and ion transmission. The Ion MAX source allows full probe adjus™ent in the x, y, and z directions so that probe position can be optimized for maximum sensitivity and robustness. Its ultra-high temperature, self-cleaning APCI probe, with ceramic heater technology, virtually eliminates sample-to-sample carryover. The Thermo TSQ Quantum Ultra’s innovative technology provides the widest linear dynamic range and lowest sensitivity limits of any commercially available triple stage quadrupole mass spectrometer. The Quantum Ultra uses large diameter hyperbolic rods that allow for significant improvements in signal to noise by running H-SRM (High-resolution Selective Reaction Monitoring).

Two ThermoElectron LTQ-Orbitrap hybrid mass spectrometers: These instruments couple a linear ion trap mass spectrometer to an Orbitrap mass analyzer via an RF-only trapping quadrupole with a curved axis. The latter injects pulsed ion beams into a rapidly changing electric field in the Orbitrap wherein they are trapped at high kinetic energies around an inner electrode. Image current detection is subsequently performed after a stable electrostatic field is achieved. Fourier transformation of the acquired transient allows wide mass range detection with high resolving power, mass accuracy, and dynamic range. These instruments operate in LC-MS mode (1 spectrum/s) with nominal mass resolving power of 60,000 and use automatic gain control to provide high-accuracy mass measurements, within 2 ppm using internal standards and within 5 ppm with external calibration. The maximum resolving power exceeds 100,000 (fwhm). Rapid, automated data-dependent capabilities enable real-time acquisition of up to three high-mass accuracy MS/MS spectra per second.

Each of the electrospray MS instruments are equipped with complimentary Dionex Ultimate 3000 series nanoflow HPLCs that enable automated data acquisition. Two of the systems provide the capability to conduct online multi-dimensional separations, such as strong cation exchange coupled to reversed-phase, popularized by the Yate’s lab MudPIT technology.

Bruker Daltronics ultraflexII MALDI TOF/TOF mass spectrometer: This instrument relies on a patented gridless ion source to ensure low down due to source fouling and possesses a state-of-the-art reflectron providing high mass resolution. This instrument is complemented by the Bruker ClinProt™ liquid handling system that enables workflow integration and MALDI target plate (AnchorChip™) loading through the Bruker software that manages all aspects of sample processing and analysis, including peptide identification. The ClinProTools™ software supports interactive inspection and comparison data sets originating from samples with different clinical diagnoses differentiating features from complex protein profiles. Additionally, the ClinProTools™ software supplies highly sophisticated mathematical algorithms for the discovery of complex biomarker pattern models. BioTools™ peptide/protein spectral interpretation software supplies functionality for the identification of individual biomarkers after MS/MS analysis. The MS/MS spectrum can be used for database searching using standard database query software tools.

Ciphergen SELDI TOF mass spectrometer: This system relies on surface-enhanced laser desorption/ionization of samples prepared on chromatographically “activated” chip surfaces (ProteinChips™) where analytes are mass analyzed by time-of-flight. The system is complemented by a Beckman Biomek 2000 automated liquid handling robot. In addition, the Ciphergen system software and integrated Biomarker Pattern Software efficiently analyzes the data and displays the output of experiments in an easy to understand format with statistical features integral to the system.

Additional MSF and research laboratory resources include a Dionex series 3000 HPLC system equipped with a UV/Vis detector for workflows that require multidimensional fractionation of proteins and/or peptides prior to MS analysis. The MSF also has a Miltenyi Mini MACS Magnetic Activated Cell Sorter, Invitrogen ZOOM gel electrophoresis systems, benchtop and Eppendorf microcentrifuges, Mettler analytical and top-loader balances, a pH meter, water baths, heating blocks, baking and microwave ovens, a Pharmacia GradiFrac automated protein purification system, a Bio-Rad Modular Electrophoresis System for protein SDS-PAGE analysis, an epi-illumination fluorescence microscope, an Applied Biosystems 5700 DNA Thermal Cycler System for automated PCR DNA/RNA amplification, equipment for DNA/RNA purification, DNA electrophoresis equipment for Southern blot analysis and sequencing, two refrigerator/freezers, a glass-front chromatography refrigerator, and a fume hood. A section of the laboratory is maintained for the purpose of secure processing, cryopreservation, and culturing of human biological samples and includes shielded areas for blood and tissue preparation, a six foot Laminar flow hood, a CO₂-enriched incubator, Sorvall benchtop centrifuges, an automated controlled-rate freezing apparatus, Coulter counter, inverted phase microscope, and refrigerator. Dedicated central facilities house -80°C freezers for long-term storage of biological specimens.

The MSF computer infrastructure comprises a 72-CPU parallel virtual machine cluster/server with 5 TB of local RAID-based storage that provides immediate mass spectral data analysis needs.