Analytical Resources

Imaging and image analysis - The use of gels (both one and two dimensional) as a key protein separation technology is recognized and the core supports analytical imaging of gels stained with either fluorescent or visible dyes.

The Fuji FLA-5000 imager is capable of high-resolution gel imaging (down to 25 μm pixel size) and can save images in standard TIFF format for downstream image analysis.  Gels as large as 40 x 56 cm can be imaged. The scanner is equipped with three lasers (630nm, 473nm, and 532nm) and a set of emission filters that allow highly sensitive detection of Sypro and CyDye fluors.  A flatbed scanner with transparency adaptor is also available for clients wishing to image Coomassie or silver stained gels.

Users have access to Phoretix Evolution, a sophisticated 2D gel analysis package produced by Nonlinear Dynamics that allows multi-gel experiments to be analyzed quickly and easily.  It also supports the analysis of multiplexed experiments where different pools of fluor-labeled proteins are separated on a single 2D gel and imaged simultaneously. 

Mass spectrometry – Four mass spectrometers are available in the core facility.

ABI/Sciex QSTAR XL

This hybrid quadrupole-TOF mass spectrometer is equipped with both PicoView (New Objective) PV400 nanosources or IonSprayer sources (MDS Sciex) for sample introduction.  The ability to collect high resolution, high mass accuracy (better than 20 ppm) MS and MS/MS data with this instrument is a key capability in performing protein identification, expression analysis, and analysis of post-translational modifications.  The mass spectrometer is capable of resolving ions from 50-6000 m/z in resolving mode and 45- 40,000 m/z in transmission only mode. Resolution in resolving mode is generally better than 8,000 FWHM (full width at half maximum peak height).

ABI/Sciex Q TRAP 4000
Multiple reaction monitoring (MRM) experiments are carried out with this instrument which contains, an advanced hybrid triple quadrupole/linear ion trap.  This instrument uses MDS SCIEX Linear Ion Trap (LIT) technology, which enables excellent quantitative and qualitative performance within a single system. Use of a quadrupole as a linear ion trap significantly enhances ion trap performance by increasing ion capacity, improving injection and trapping efficiencies, and increasing duty cycle.  This system provides true triple quadrupole MRM at the highest level of sensitivity, as well as extended dynamic range, ensuring superior quantitation performance for peptides.  The Q TRAP can be configured with either the TurboIonSpray source or the MicroIon source, mounted on the MDS Sciex nanosource. 

ThermoFinnigan LTQ

The LTQ quadrupole linear ion trap instrument is a highly sensitive ThermoFinnigan ion trap. The LTQ confines trapped ions in a 2-dimensional space instead of 3-dimensional one, allowing confinement of a greater number of ions without degrading resolution and mass accuracy due to space charging effects. The instrument is also capable of scanning at much higher rates than many other instruments, typically producing over 15,000 scans during a single LC-MS experiment.  For highest sensitivity the instrument is also fitted with a ThermoFinnigan IonMax nanospary source and packed tip probe.

ThermoFinnigan LCQ ion trap with an atmospheric MALDI source

The LCQ ion trap instrument is fitted with an atmospheric pressure MALDI source from Mass Technologies for rapid collection of MS/MS spectra from unfractionated protein digests.  This system can analyze proteins from approximately 100 gell bands in a day, and is especially useful for samples produced from gel digests where sample complexity is not great.  This method is more cost effective since it requires less instrument time than LC-MS analysis (which would be 100 hours for 100 gel bands); however it is also less sensitive than LC-MS and is usually only used when there is an abundance of material to work with (i.e., greater than 100fm of a pure protein).

MS/MS Data analysis – A wide range of data analysis tools are supported in the core.  Software for interpreting MS/MS spectra and identifying peptides include Sequest, Mascot, and X!Tandem.  Scaffold software from Proteome Software is used to validate MS/MS based peptide and protein identifications. Peptide and protein identifications are evaluated by this software which provides a statistical analysis of reported identifications.  The software also produces output files that can be easily shared with users via the free Scaffold viewer.