Equipment

The major equipment in the Proteomics Core can be grouped by the services that require them.

Protein identification and differential proteomics

  • Thermo Scientific LTQ Orbitrap. The workhorse instruments for services involving protein identification and differential proteomics projects are four Thermo Scientific LTQ Orbitrap mass spectrometers. These are hybrid mass spectrometers composed of a linear ion trap coupled to an Orbitrap mass spectrometer.

    The LTQ Orbitrap has 60,000 to 100,000 resolution and less than 2 parts per million (ppm) mass accuracy. The speed, dynamic range and sensitivity of this instrument make it ideal for proteomics experiments.

    One of the core's LTQ Orbitraps is equipped to perform electron transfer dissociation (ETD). This enhancement allows for fragmentation of the peptide and keeps the labile post-translational modifications intact for identification. All four LTQ Orbitraps are configured with Eksigent NanoLC-2D solvent delivery systems and autosamplers.

  • Hardware. Comprehensive hardware is necessary to analyze and store the data generated from the Oribitraps. This includes a Dell M1000e blade server enclosure populated with 16 blades: 12x M605 dual quad-core Opteron 2378 (2.4Ghz, 32GB RAM) and 4x M710HD dual six-core Xeon X5670 (2.93Ghz, 48GB RAM).

    This enclosure is attached to a Dell EqualLogic PS6010X iSCSI SAN storage array with 16x 600GB 10k SAS drives. This hardware runs multiple protein search engines in an automated workflow — orchestrated by software called Swift, which was developed in-house &madsh; as well as database and Web servers for data storage and presentation.

Mass determination

  • Agilent LC/MSD time-of-flight. The primary use of the Proteomic Core's Agilent LC/MSD time-of-flight mass spectrometer is for mass measurements of intact peptides and proteins. This instrument has a mass resolving power of 10,000 and mass accuracy better than 5 ppm for internally calibrated spectra.

Peptide synthesis

  • Apex 396. For small-scale (0.04 millimole, or 10 mg or less) synthesis requests, the Proteomics Core has two Apex 396 multiple peptide synthesizers for parallel synthesis of peptides. These instruments are capable of synthesizing up to 48 peptide sequences simultaneously by Fmoc solid-phase peptide chemistry.
  • CEM Liberty. For large-scale (10 millimole or 20-40 mg, and 25 millimole or greater than 40 mg) synthesis requests, the core's three CEM Liberty 12-channel automated peptide synthesizers are used. These synthesizers are capable of sequentially synthesizing up to 12 different peptides automatically using either Fmoc or Boc solid-phase chemistry.

Protein purification

  • Bio-Rad BioLogic DuoFlow Pathfinder 20 and GE ÄKTAexplorer 100. Protein purifications by fast-performance liquid chromatography are achieved using either one of two BioLogic DuoFlow Pathfinder 20 systems or a ÄKTAexplorer 100 system.

    These versatile instruments are capable of handling a wide variety of separation techniques, sample sizes and column dimensions.

Electrophoresis

  • Bio-Rad and Invitrogen systems. The Proteomics Core has both Bio-Rad and Invitrogen electrophoresis systems. Mini-, midi- (1-D and 2-D) and large- (1-D and 2-D) format gels can be accommodated.

The Proteomics Core has three instruments for capturing gel data:

  • Bio-Rad GS-800. The Bio-Rad GS-800 densitometer gives high-resolution 12-bit images of visible light stains (for example, Coomassie and silver).
  • Bio-Rad Molecular Imager FX. The Bio-Rad Molecular Imager FX is a laser scanner for obtaining high-resolution 16-bit fluorescent images at 488, 532 or 635 nanometers.
  • ProteinSimple FluorChem M. The ProteinSimple FluorChem M is a cooled 8-megapixel charge-coupled device (CCD) camera imager capable of electrochemiluminescence (ECL) capture from Western blots and acquisition of visible and fluorescent images.

For analyzing electrophoresis data, the Proteomics Core offers:

  • Software. Available software includes Bio-Rad PDQuest software for 2-D gel analysis, and Bio-Rad Quantity One and ProteinSimple AlphaView for densitometry measurements.

Nanofluidic immunoassay

  • ProteinSimple CB1000. The Proteomics Core has a ProteinSimple CB1000 for assays that explore, characterize and quantify cell-signaling proteins. The instrument uses capillary-based isoelectric focusing to separate and resolve various protein phosphorylation states.