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University of Kentucky Proteomics Core Facility

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Two New Mass Spectrometers at UK Proteomics Core (Updated January 2011)

LTQ Velos Orbitrap with ETD: Ideal for identifying low abundance proteins and characterizing protein post-translational modifications.

LTQ Velos Orbitrap with ETD

 

 

 

 

 

 

 

Highlights:

  • Superior sensitivity at the sub-femtomole (< 1E-15 moles) level.
  • ETD (Electron transfer dissociation) - especially valuable for characterizing protein post-translational modifications.
  • Front End: a dedicated Eksitgent NanoLC-Ultra and NanoFlex ChipLC system.
  • Non-splitting flow control
  • Outstanding reproducibility
    Outstanding mass accuracy (routinely 1-2 ppm) and resolution (up to 100,000).
  • Microfluidic chips as columns

Front End: a dedicated Eksigent NanoLC-Ultra and NanoFlex ChipLC system.
Non-splitting flow control
Outstanding reproducibility
Microfluidic chips as columns

TSQ Vantage Triple Quadrupole (installed January 2011): Workhorse of quantifying proteins, peptides and metabolites, i.e. biomarker validation & pharmacokinetics.

TSQ Vantage Triple Quadropole (installed January 2011)

Highlights:
Linear dynamic range of four orders of magnitude for quantitation (0.01fmol-100fmol).
Multiple scanning modes: High-sensitivity full-scan, Selected Ion Monitoring (SIM). Selected Reaction Monitoring (SRM) and others.
Quantitation-Enhanced Data-Dependent MS/MS (QED-MS/MS) for simultaneous compound confirmation and quantitation

Versatile HPLC front end:
Conventional flow with Shimadzu HPLC and electrospray (HESI-II) source.
Capillary and Nano-flow with Eksigent HPLC and nanospray source.

Two Existing Mass Spectrometers

4800 MALDI-TOF/TOF (installed August 2007): High throughput protein identification.

4800 MALDI-TOF/TOF (installed August 2007)

 

  • Highlights:
    High throughput proteomics analysis.
    MALDI source
    Low femto-mole sensitivity
    Routinely 10-20 ppm mass accuracy
    HPLC-MALDI separation available.

QSTAR XL Quadrupole Time-of-flight (installed December 2002)

Highlights:
Regular and Nano-ESI sources.
Direct infusion or HPLC front end separation.
Users can be trained to operate if regular usage is needed.
Questions? Contact Dr. Haining Zhu (haining@uky.edu) or Dr. Carol Beach.

 

 gel apparatus

Bio-Rad Spot Picker:   SDS PAGE is often the final step in protein purification before submission of samples for protein identification.  The core facility has access to a Bio-Rad spot picker which can create an image of visibly-stained gels or Sypro Ruby fluorescent dye-stained gels.  Spots or bands of interest are designated for excision by a robot which will remove a 1.5 mm core from the gel and deposit the gel core in a 96-well microtiter plate.  The enclosure around the robot acts to reduce keratin contamination and also provides protection when fluorescently-stained gels are exposed to UV light. 

 Typhoon apparatus

Typhoon 9400 Phosphorimager/Scanner:  The core facility has an Amersham Typhoon 9400 Phosphorimager/Fluorescence Scanner.  This versatile system will image gel sandwiches, gels, membranes, microplates and even microarrays, and it provides high sensitivity detection and quantitation of fluorescent stains and probes used for gel analyses of proteins as well as more traditional immunobloting procedures.  Automated four-color fluorescence scanning allows multiplexing of multiple targets in the same sample, ensuring accuracy of analysis, increasing throughput, and saving time.  This system can be used with the DIGE (Difference Gel Electrophoresis) methodology which uses amine-directed cyanine-based dyes (Cy-Dyes) to label proteins for differential quantitation on a single gel. 

PDQuest Gel Analysis Software:  The core facility has a site license for the Bio-Rad gel analysis software PDQuest.    This imaging software helps to align multiple gels and to organize the array of spots so that each spot can be individually analyzed. There is also a vertical and horizontal streak reduction routine.  3D data (X-, Y- position and fluorescence intensity) can be used to calculate a volume for each spot that can be used to quantify the amount of protein in the spot and to compare it to other spots. The spotís volume can be normalized to the total spot intensities to take into account loading variation.  This allows spots to be compared on different gels.   Once normalized, data from several gels can be averaged and used to generate statistically significant databases of spot volume and, therefore, amount of protein in the spot.  These databases can then be compared to detect differences in protein levels in specific spots, so this approach is a visual method of expression difference quantitation.