Resources

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.

Mass Spectrometry

Four mass spectrometers are available in the core facility.

 

Thermo Scientific LTQ

Analytical-Resources-LTQThe 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.

 

 

Thermo Scientific Velos

VelosThe 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.

 

 

Thermo Scientific Orbitrap Fusion

Orbitrap FusionIn additional to increased sensitivity over previous generations of mass spectrometers the Orbitrap Fusion has much higher mass accuracy than any other instrument in the Shared Resource; not only giving a researcher the ability to detect minute mass differences caused by a neutron's association with different nuclei, but doing so in an instrument that fits nicely on a table top. The instrument is currently being used to support 10-plex TMT and SILAC based quantitative proteomics, targeted analysis of peptides using high-resolution fragment ion spectra, ETD enabled identification of disulfide bonding and chemical crosslinks in proteins, and analysis of post-translational modifications. The instrument is also interfaced with a LEAP PAL robot to support automated hydrogen/deuterium exchange experiments."

 

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 Comet, 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 targeted peptide analysis  we utilize Skyline software developed by Brendan MacLean in 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 also use Byonic from Protein Metrics. This software has an unrestricted PTM search option, which allows the identification of unknown post-translational modifications and glycoslyation sites. Additional supported software includes MaxQuant, which is used to process SILAC results from the Orbitrap Fusion."

 

Other Instrumentation

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.