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.
Front End: a dedicated Eksigent NanoLC-Ultra and NanoFlex ChipLC system.
TSQ Vantage Triple Quadrupole (installed January 2011): Workhorse of quantifying proteins, peptides and metabolites, i.e. biomarker validation & pharmacokinetics.
Versatile HPLC front end:
Two Existing Mass Spectrometers
4800 MALDI-TOF/TOF (installed August 2007): High throughput protein identification.
QSTAR XL Quadrupole Time-of-flight (installed December 2002)
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.
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.
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