University of Kentucky Proteomics Core Facility

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Ciphergen system:    The core facility contains a Ciphergen PBSIIc Biomarker Discovery System which is a linear MALDI-TOF mass spectrometer capable of analyzing samples bound to specialized Ciphergen targets.  The Ciphergen targets are chemically derivatized (reverse phase, normal phase, weak cation exchange, strong anion exchange, immobilized metal affinity, and, for covalent immobilization, acyl imidazole and epoxy reactive groups) and bind proteins, peptides, and other biomolecules which can then be analyzed by mass spectrometry.  This type of analysis has been named “SELDI” (Surface Enhanced Laser Desorption Ionization).  The chemically derivatized surfaces can act to reduce sample complexity since not all proteins are bound to a particular target.  Thus particular proteins are selectively bound (depending on the binding conditions employed) and unbound proteins are washed away.  The targets are then analyzed in the mass spectrometer.  The profile of masses is relatively quantitative and reproducible. This is a medium- to high-throughput system and spectra are automatically posted to the server which can be accessed by users for data analysis using the Ciphergen Express software package.  Kelly Conrads of Ciphergen produced a Powerpoint presentation which describes the capabilities of the SELDI system. A drawback of the system is that it is not capable of fragmenting peptides (MS/MS) for protein identification, however, its strength is providing a profile of proteins that can be used to detect differences; for example, between a normal and disease state, or between one treatment versus another.  To identify a peak of interest its identification needs to be determined by some other technique.

QStar XL QTOF mass spectrometer:    The core facility also uses an Applied Biosystems QStar QTOF mass spectrometer.  This is a high resolution hybrid machine which can accurately measure peptide masses and confine and fragment peptides in a collision cell so that the fragment masses may be measured.  These MS/MS data are used in conjunction with unfragmented peptide masses to identify proteins (see Protein Identification). Peptide mixtures are separated before entering the mass spectrometer by reverse phase HPLC using an Eksigent nanoLC system.

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MALDI TOF/TOF Mass Spectrometer:  We are pleased to announce the installation of our newest instrument, an Applied Biosystems 4800 Plus MALDI TOF/TOF mass spectrometer.  The MALDI TOF/TOF complements the QStar capabilities by using a MALDI source instead of an electrospray source.  Both machines are capable of isolating and fragmenting peptides for protein identification.  Electrospray ionizes samples in liquid and is suited for “on the fly” analysis of eluent from an HPLC column.  MALDI ionizes dry samples which have been co-crystallized with a laser light-absorbing matrix and has been called “the ultimate stop-flow technology”.  Interesting spectral peaks may be chosen (at a later time) for fragmentation (MS/MS) yielding peptide sequence ions, so there is time for making decisions about further analysis.  

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 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 spot’s 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.