Proteomics Research Center Facilities
Laboratories
Mass Spectrometry Instrument Laboratory
2,414 sq. ft. space, climate controlled to within ± 2°C. It houses all mass spectrometry systems, Tecan robotics system, an LC Packings HPLC system, and a Dell workstation. Room is plumbed for house nitrogen (UHP boil off LN2 Dewar). Several 4°C refrigerators are in this building. The lab space is shared between large- and small-molecule mass spectrometry research.
Mass Spectrometry Support Laboratory
374 sq. ft. climate-controlled space. It houses a Sutter Instrument P-2000 laser capillary puller, a Beckman DU 800 UV/Vis spectrophotometer, and ample bench space for sample preparation. Water service and hookups to house nitrogen are available; several -20°C freezers and 4°C refrigerators are in this area.
Wet Chemistry Laboratory
587.5 sq. ft. climate-controlled space for general protein chemistry support. The lab is dedicated to supporting the mass spectrometry research program. Multiple power points (120V and 220V) are available. Major fixtures include a chromatography refrigerator (on backup power); two conventional chemical fume hoods; a Labconco biosafety laminar flow hood for tissue culture, microbiological, or cleanroom work; a Savant centrifugal evaporator; pH meters; water baths; heated microcentrifuge tube shakers; a large floor shaker for incubation and agitation of large quantities of material; an Amersham Biosciences FPLC (described below); and a Beckman Allegra 25. Ample glass, plastic ware, and other general laboratory equipment are available for use. A Barnsted Nanopure (18MΩ) water system is available for ultrapure water needs.
Equipment
Typhoon Trio Variable Mode Imager
A variable mode imager that produces digital images of radioactive, fluorescent, and chemilluminescent samples with a 25-micron minimal pixel size. This instrument is used for imaging Cy2-, Cy3-, and Cy5-labeled proteins, as well as Sypro Ruby-stained proteins, all key components of the DIGE platform.
Dell Precision 670 Workstation
A computer workstation for DIGE image and proteomic statistical analysis. Components consist of an Intel Xeon 3.2 GHz processor, 4 GB DDR2 SDRAM at 400 MHz, a 1 TB RAID5 SCSI array, and dual 19-inch LCD displays. Software installed consists of Decyder 2D (version 6.5), Decyder Extended Data Analysis (version 6.5), Oracle 10g for data organization and backup, and Spotfire DecisonSite for Functional Genomics and Statistics (version 8.1).
Applied Biosystems Proteomics 4700 Tandem MALDI TOF/TOF Mass Spectrometer
A high-performance MALDI mass spectrometer with true tandem capabilities. Equipped with 200 Hz Nd:Yag laser, ultimate mass resolution to 30,000 at m/z 12,000 Da, sensitivity quoted at 10 amol of a single peptide analyte. Can accept up to three collision gases, integrated Oracle database for storage of results, capable of fully automated operation; data-dependent MS/MS analyses can be performed. Analysis is automated using a plate-loader with a capacity of up to 24 plates. The autoloader has a built-in bar code reader to allow rapid plate-to-plate transfer. This integrated system runs on the GPS Explorer software platform for data management.
Thermo Electron LTQ Linear Ion Trap Mass Spectrometer
A linear ion trap mass spectrometer capable of advanced scanning modes, including data dependent scans, ion mapping, neutral ion mapping, parent ion mapping, user-selectable dynamic exclusion, Nth-order triple play, data-dependent zoom map, data-dependent ion tree and total ion map, custom data dependence, real-time modifications, and Nth-order neutral loss data dependence. Equipped with conventional ESI probe with orthogonal spray. New Objective Picoview nanospray source is also available for static and dynamic (LC) nanospray experiments. Mass range to 20,000 Da, normal operating ranges at 2,000 and 4,000 Da. Integrated Xcalibur data system allows rapid programming of data-dependent LC-MS/MS experiments. Control of LC is available from Xcalibur using a software plug-in.
Thermo Electron LTQ ETD Linear Ion Trap Mass Spectrometer
A linear ion trap mass spectrometer capable of ion-ion reactions for electron transfer or proton transfer. This system is equipped with a second (negative ion chemical ionization) ion source to generate counter ions for reaction with oppositely charged ions generated by electrospray. The system is controlled and programmed using ion trap control language (ITCL). It is used for instrument development and fundamental research in proteomics.
Thermo Electron LCQ-Deca-XP Ion Trap Mass Spectrometer
A high-performance quadrupole ion trap mass spectrometer for electrospray MS analyses. Equipped with conventional ESI probe with orthogonal spray. New Objective Picoview nanospray source is also available for static and dynamic (LC) nanospray experiments. Mass range to 20,000 Da, normal operating ranges at 2,000 and 4,000 Da. Integrated Xcalibur data system allows rapid programming of data-dependent LC-MS/MS experiments. Control of LC is available from Xcalibur using a software plug-in.
Agilent 1100 Series Nanoflow LC System
Two dual-piston in series, with servo-controlled variable stroke drive, floating piston, active inlet valve, solvent selection valve, and electronic flow control for flow rates from 0.1 to 1 µl/min. Contains a thermostated microwell plate autosampler capable of injection volumes as lower l. Sample capacity for the unit is two well plates (96 and 384), 10?than two additional 2-ml vials, or 100 × 2-ml vials in a standard tray. Unit is also equipped with a micro vacuum degasser and series control module. A second standard 100 series pump is used for 2-D LC applications and automated sample loading.
Amersham Biosciences AKTA Purifier FPLC
An integrated high- and medium-pressure liquid chromatography system, capable of pressures to 25 MPa and flow rates up to 10 ml/min. Dual wavelength UV/Vis detector, sample injection up to 2 ml and integrated fraction collector capable of handling conventional test tubes, microcentrifuge tubes and microtitre plate formats. All analyses are under data system control using the Unicorn software package.
Dionex LC Packings Ultimate Nano HPLC System, Switchos Column Switching Unit, Famos HPLC Autosampler
Easily configurable for 75, 300, or 1,000 µm column use, it can deliver reproducible gradients at 200 nl/min, two automated 10-port valves for automated column switching, preconcentration or 2-D chromatography experiments, autosampler capable of 1 µl injections, all components under control of Xcalibur mass spectrometry data system using a plug-in package.
Dionex LC Packings Probot Microfraction Collector
A microfraction collector capable of collecting effluent as small as 7 nL onto MALDI targets for LC-MALDI analyses under full computer control.
Eksigent NanoLC 2D System
This system is interfaced to the LTQ ion-ion reaction instrument. Optimized for ultra-low flow rates of first dimension 1-20 µL/min, second dimension 50-1000 nL/min. Includes two independent binary gradient pump systems with a 10-port column switching valve accommodating two trap columns and an analytical column, and an integrated temperature-controlled autosampler with 6-port bio-compatible sample injection valve. Dead volume for the first dimension 300 nL from mixer to column (low-flow dimension 65 nL from mixer to column). The autosampler has either 96- or 384-well plate capacity. Peltier-type temperature-maintained sample compartment, 5–40°C; cooling capacity of 12°C below ambient. Dual-needle design for seal piercing. Integral 6-port bio-compatible injection valve.
Sutter Instrument P-2000 Laser Capillary Puller
A laser capillary puller for rapid production of nanospray tips and integrated nano LC-nanospray tip devices from fused silica. Instrument is capable of pulling a 1-5 µm capillary orifice reproducibly.
Beckman Coulter Allegra 25R Centrifuge
Capable of speeds up to 25,000 Gs, integrated cooling feature, fixed angle and swinging bucket rotors available. IEC B-22M floor model high-speed refrigerated centrifuge (maximum rcf of 49,300 x G @ 21,000 rpm). Sorvall RC-5B floor model refrigerated superspeed centrifuge (same rcf/rpm specifications as IEC).
Ettan IPGphor II Isoelectric Focusing Unit
This is used for the first dimension of shotgun proteomics experiments. Can be used to focus 12 IPG strips using the manifold or 12-strip holders. The unit has Peltier solid-state temperature control (18-25 °C) and has a programmable controller for voltage, temperature, and time.
Gel Systems
Two Ettan IPGphore II (Amersham; 1st dimension isoelectric focusing units); can focus 12 IPG dry strips in a run. Two Multiphor II (Amersham; 2nd dimension analytical units) with thermostatic circulator and power supply; each unit can run on 110 x 250 x 0.5 mm gel. Three Ettan DALT units (Amersham, 2nd dimension large format) with power supply, each unit can handle twelve 155 x 196 x l mm gels in one run. Gel costing equipment (large format) will be used for the majority of gels produced in-house.
Visualization System
Hoefer Auto Stainer, used for silver staining, holds one DALT or two analytical gels; Infinity LS Shaker, platform orbital shaker (3-100 rpm), used for Sypro Ruby and CCB staining, can process at least 12 gels simultaneously. Labscan Image Scanner (Amersham); white light imager; VersaDoc Imaging System (Bio-Rad), image-stained gels with white or UV light, CCD camera; PDQuest (Version 7.1) image analysis software (Bio-Rad).
Spot Picker
Ettan Spot Picker (Amersham) automatically picks selected protein spots from stained gels.
Protein Digestion System Genesis Proteome Robotic Digestion Station
Integrated, flexible liquid handling robotics package for automated tryptic digestion and other assays as needed. Liquid handling arm equipped with four fixed liquid-sensing dispenser tips and four removable tips. Robotic manipulator arm for moving samples around the work table. Fully programmable using flexible Genesis programming language. Custom-built sample carriers with Peltier cooling. MALDI sample spotting fully integrated with the digestion process.
Autoclave
A floor model Beta Star Corporation Laboratory steam sterilizer and a table-top VWR unit.
Well-Former Robot for Automated Peptide IPG Strip Extractor
This modified Gilson robotics system uses its liquid-handling hardware to automate the IPG-IEF strip extraction for the first dimension of shotgun proteomics. It is a test unit developed as part of the commercialization of our proteomics platform by GE Healthcare. It is capable of extracting eight strips simultaneously and can deposit extracts in separate 96-well plates. Strips are extracted in 3-mm sections.