Imaging and Image Analysis
PSR has an Epson Expression 1600 scanner with a recessed scan bed that is ideal for imaging gels stained with visible dyes, such as Coomassie or Imperial Blue. We no longer support the imaging of florescent dyes at PSR. However if you have a gel that you need a florescence imager for let us know, and we can point you to other resources at OHSU.
Four mass spectrometers are available in the core facility.
Applied Biosystems QTRAP 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.
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 nanospray source and packed tip probe.
The Velos is Thermofinnigan's latest linear ion trap. It features improved ion optics, a dual pressure ion trap, and faster scan speeds then the older LTQ models. There are currently two Velos mass spectrometers in PSR. One of the instruments is also outfitted to do Electron Transfer Dissociation, or ETD. The ETD source gives the shared resource more options to explore post-translational modifications such as phosphorylation or di-sulfide bonding.
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 3.0 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.
We also utilize the PAW Pipeline developed here at OHSU by Phil Wilmarth. PAW gives superior sensitivity and quantitative options. This is especially true for complex mixtures of protein and larger datasets. It also allows us to tailor the statistical analysis to meet the needs of the client, and has the ability to produce more accurate lists of peptides containing post-translational modifications.
For MRM analysis on the QTRAP 4000 we utilize Skyline developed by the MacCoss Lab at the University of Washington. Skyline gives us the ability to develop and refine methods for SRM/MRM analysis and quantify the resulting data.
We've recently acquired Byonic from Protein Metrics. This software has an unrestricted PTM search option, which allows the identification of unknown post-translational modifications and glycoslyation sites. We've also acquired MaxQuant, a search engine designed for high mass accuracy datasets, in anticipation of the arrival of our new Orbitrap mass spectrometer.
The Proteomics Shared Resource also has a wide variety of other instrumentation as well. Notable pieces include a Biotek Epoch Microplate Spectrophotometer which is mainly used for protein assays, a Surveyor HPLC for chromatographic separations, a Jouan BR4i centrifuge, and two vacuum concentrators.