Protein Identification and Localization Core

Equipment

Protein Identification Component

The Proteomics Core occupies approximately 2,000 square feet at the University of Michigan.

Major equipment at the University of Michigan site includes:

1. Three Applied Biosystems model 4800 MALDI TOF/TOF mass spectrometers for proteome mapping, identification of post-translational modifications, and iTRAQ quantitation with 10 ppm mass accuracy and sensitivity to at least one femtomole.
2. ThermoFisher Orbitrap with an Advion Nanomate interface that improves reproducibility and sensitivity for phosphoproteome analyses.
3. Virgen InstrumentsMiniTOF for peptide mass fingerprinting, development, and QC
4. Two Agilent HPLC with MALDI fraction collector for interfacing LC with the TOFTO
5. Eksigent capillary HPLC with non-ferrous solvent path coupled to the Orbitrap
6. Michrom HPLC for SCX separations
7. Sample handling robot to support 2D gels
8. Laser and transmittance scanners for imaging 2D gels
9. 2D gel apparati with capacity for up to 40 gels per day
10. Off-gel electrophoresis system for preparative isoelectric focusing.

The Proteomics Core has a complete IT infrastructure and a full-time systems administrator to manage several Linux and Windows servers, and several PC and Solaris workstations. The laboratory maintains software for image analysis and an extensive system for information management. The Proteome Consortium also operates a 6 cpu NT server, and several NT servers for administrative and database search tools. The Consortium has site licenses for Mascot, SeQuest, Protein Pilot, Protein Prospector, and Protein Global Server and maintains an X!Tandem (3) server and an eight cpu Linux cluster. Other software available includes Scaffold Q+, PEAKS, MaxQuant, and the TransProteome Pipeline. Additional Linux cluster support will be provided by the Michigan Center for Biological Information as needed. Data dissemination by the core will be through Tranche (proteomecommons.org/tranche) while project management and annotation will be managed through proteomecommons.org. Both sites are developed and managed by the Andrews laboratory.

Protein Localization Component

Please be sure to submit this request form to be eligible to receive the microscope subsidy.

Major microscopes available to GI Center users for protein localization and cell imaging on a recharge basis include

1. Olympus FluoView 500 Laser Scanning Confocal Microscope
2. Zeiss LSM 510-META Laser Scanning Confocal Microscope
3. Nikon widefield inverted stage microscope equipped for FRET analysis
4. Phillips CM-100 Transmission Electron Microscopy (TEM)
5. Ancillary Equipment

1.   Olympus FluoView 500 Laser Scanning Confocal Microscope is controlled by a 2.4 Ghz personal computer under Windows 2000 and is capable of imaging 5 separate channels simultaneously (4 fluorescence + 1 transmitted light photomultiplier detectors) offering highly efficient, maximum emission sensitivity and the ability to record scanned images in 12 bits or 4096 gray levels, thus allowing quantitative linear measurement of fluorescence within regions of low contrast as well as very high contrast. Users are able to image a wide variety of fluorophores with laser excitation that includes Blue Violet (405 nm), Multi-Line Argon Blue (458,488,515nm), Helium Neon Green (543nm) and Helium Neon Red (633nm) for standard Blue, Green, Red and Far-Red fluorochromes. The FV500’s acoustical optical tuning filter (AOTF) and adjustable scan speeds provides for minimal specimen fading, sequential scanning for reduced fluorescence cross talk, multiple regions of excitation, high resolution imaging (up to 2048 x 2048 pixels) of fixed or static samples, and rapid recording of kinetic events. Optical sections in the z plane can be collected using a step motor attached to the fine focus control of the microscope and driven by Fluoview software. The system is also equipped with Differential Interference Contrast (DIC) objectives and condensers and has the ability to capture transmitted light images with a highly sensitive photomultiplier (PMT) transmission dector. A similar microscope with slightly different peripherals but running under the identical operating system is available for use in the MIL.

The Images can be saved to a peripheral hard drive for later analysis. Integral software allows for analysis of saved images in 2 dimensions (e.g., brightness vs. time); confocal images obtained in a “z” series can be volume rendered and analyzed in 3 dimensions. Data can be archived on CD, DVD, or Zip disks, or transferred to an alternate image analysis platform with greater storage capacity. (Microscopy Service Request Form)

2.  Zeiss LSM 510-META Laser Scanning Confocol Microscope
This micropscope is mounted on a Zeiss Axiovert 100M inverted microscope is located in the MIL. This instrument is equipped with four lasers offering a wide range of excitation wavelengths. Coherent Enterprise laser for UV (351,364 nm), Argon laser for FITC/GFP (458, 488, 514 nm), Helium Neon 1 laser for Rhodamine, Texas Red, Cy3 (543 nm), and Helium Neon 2 laser for Cy5 (633 nm). Using the META system, the researcher can easily separate highly overlapping emission peaks. (Microscopy Service Request Form)

3.  Nikon widefield inverted stage microscope equipped for FRET analysis
This instrument is located in the MDRTC Core. It is equipped with specialized Chroma CFP and YFP excitations/emission filters, Sutter excitation and emission filter wheels and controller (Lambda 10-2), a Prior automated x, y, z stage, a TE-ICV incubator with digital thermistor probe and incubator case, and a Hamamatsu ORCA extended range digital CCC camera (C4742-95-12ER). This camera rapidly captures images at rates ranging from 8.3-45 frames per second with very high quantum efficiency resulting in shorter exposures of sensitive samples to fluorescent light. Thus, the ORCA digital camera allows us to get the maximum performance and utility from FRET microscopy. The acquisition and analysis of FRET data is semi-automated with the use of specialized Metamorph macros/journals. This multiwavelength fluorescence workstation is also equipped with appropriate filters and software (Metafluor) to work with fluorescent probes such as fura-2 for monitoring intracellular calcium concentrations. (Microscopy Service Request Form)

4.  Phillips CM-100 Transmission Electron Microscopy (TEM)
The Phillips CM-100 TEM is maintained and operated in the Morphology and Image Analysis Laboratory (MIL), part of the Department of Cell and Developmental Biology. It is equipped with a motorized stage and a Kodak 1.6 megaplus digital camera capable of capturing electron images directly from the viewing screen. This microscope will be used for the collection of digital EM images and for the direct transfer of data from the microscope to the image analysis software reducing the need for photographic film, chemicals, and paper. Use of this equipment is available to all University of Michigan investigators on a recharge basis for $45/hour. (Microscopy Service Request Form)

5.  Ancillary Equipment
Other equipment available in the MIAC or Dr. Ernst’s laboratory include two cryostats, a RMC MT-7 ultramicrotome and CR-21 cryosectioning attachment, knifemakers and a Kodak Digital Sciences 865OPS dye sublimation printer.