The OHSU Protemics Shared Resource facility was established to make state-of-the-art mass spectrometry based protein analysis analytical capabilities available to the biomedical research community at OHSU.
Klimek, John E., B.S.
Role: Research Associate
McMillen, Debra, M.S.
Reddy, Ashok, Ph.D.
Role: Associate director
Wilmarth, Phillip, Ph.D.
Laboratory has two of these instruments.
Two of these systems are available.
"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. This instrument does not have a Electron Transfer Dissociation (ETD) source.
"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. This instrument 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.
Laboratory has two of these system available for use.
"All Reagents part of iTRAQ kit from Applied Biosystems Cat # 4352135, except Trypsin."
"Sample preparation is one of the often overlooked steps in achieving high quality results in a proteomics experiment. Despite the enormous technical advances in mass spectrometric instrumentation and software, contaminated samples can easily overwhelm the most advanced mass spectrometer and extremely bad samples can actually render a mass spectrometer unusable for an extended period of time."
"The PAW Pipeline was developed by Phil Wilmarth of OHSU as an improvement over previous open source protein identification tools; the initial publication can be found here. This software package provides PSR with customizable state-of-the-art protein identification software, and has significantly increased sensitivity in larger and more complex datasets."
* SCX Cartridge Protocol
* OMIX C18 Cleanup Procedure
* MCX Cleanup Procedure
* Sep Pak Cleanup Procedure
Includes protocols for:
* In-gel digestion procedure
* Small-scale solution digestion procedure
* Large-scale solution digestion procedure
An overview of mass spectrometry based proteomics.
"Now updated for version 3.0; an unofficial guide for installing and using the free scaffold viewer for mass spec results."
"This document goes over some of the sample preparation concerns unique to the Co-IP experiment. It also summarizes PSR's procedures for processing the samples, gives pointers for assessing final data from the experiment, and lists some approximate costs."
"Whole mass determination of a protein is often a relatively straight-forward analysis. When the protein is introduced into the mass spectrometer a series of charge states are produced. De-convolution software can interpret this complex spectrum, and reconstruct the mass of the protein.
Depending on the nature of the experiment a hypothetical sequence or other information may be required. Samples for this kind of analysis are usually submitted as either a liquid solution, or lyophilized powder."
"Relative Quantitation of the same protein in different samples is usually performed via spectral counting in our laboratory. This type of analysis is simple and easy to perform, as the number of times MS/MS spectra matches to a protein entry in the database has been shown to be roughly proportional to the amount of protein present.
Though the analysis of this data is relatively straight forward it requires that more effort is spent on preparing the samples. Errors introduced in the sample preparation phase can result in serious problems with the analysis.
Samples for this kind of analysis are usually submitted as either a liquid solution, or lyophilized powder.
Single gel bands are not generally compatible with the procedure however, as the counting statistics are affected by the nature of the sample. In this case the abundance difference apparent on gel is a more useful then the spectral counting numbers."
"This type of analysis is a step beyond simple protein identification. Post-Translational Modifications (PTMs) that result in a simple mass addition to the peptide can be detected using the software present in our lab. A good example of this type of modification is phosphorylation. The mass shift of the potential PTM is entered into the protein identification software, and the software performs multiple searches of potentially modified peptides looking for forms both with and without the PTM.
This analysis can take considerably longer because the search time increases geometrically with each potential modification added. Also the algorithms for identifying PTMs are less well developed then those for protein identification, meaning every potential PTM must be verified by hand! This adds considerably to the amount of time it takes to analyze the data, and drives up the overall cost of the experiment.
As with protein identification, samples can be submitted as either a liquid solution of the protein(s), or as a band from a gel. Liquid samples may also by lyophilized or taken to dryness before submission."
"Identification of unknown proteins is the bread-and-butter analysis for a Proteomics Facility. Proteins are enzymatically digested into peptides, which are then introduced into a mass spectrometer via a liquid chromatography system. Once inside the mass spectrometer we fragment the peptides to produce MS/MS spectra. These fragmentation patterns are matched against theoretical spectra from a protein database. Scoring algorithms and statistical tools are then used to determine the identity of the proteins in the sample.
Samples can be submitted as either a liquid solution of the protein(s), or as a band from a gel. Liquid samples may also by lyophilized or taken to dryness before submission."
"If you'd like to attempt an SRM experiment, close consultation with the Proteomics Shared Resource is necessary. These experiments usually take significant amounts of time and resources, but in the end can yield a very nice dataset. If you'd like to perform this type of analysis please contact PSR, so that we can assist you in this process."
This software collects and analyzes mass spectrometry data.
"Mascot is a powerful search engine which uses mass spectrometry data to identify proteins from primary sequence databases. While a number of similar programs available, Mascot is unique in that it integrates all of the proven methods of searching. These different search methods can be categorised as follows:
* Peptide Mass Fingerprint in which the only experimental data are peptide mass values.
* Sequence Query in which peptide mass data are combined with amino acid sequence and composition information. A super-set of a sequence tag query.
* MS/MS Ion Search using uninterpreted MS/MS data from one or more peptides."
"A comprehensive and expandable software platform for the analysis of quantitative and qualitative proteomics data."
This pipeline performs protein probability calculations.
"The PAW Pipeline was developed by Phil Wilmarth of OHSU as an improvement over previous open source protein identification tools. This software package provides PSR with customizable state-of-the-art protein identification software, and has significantly increased sensitivity in larger and more complex datasets."
"Scaffold has proven its performance in over 300 academic, public and private research facilities around the world with the ability to display a visual comparison of the proteins found by multiple search engines across all the samples of an experiment." "* Compare multiple samples and categories with advanced statistical analysis.
* Identify proteins by validating MS/MS data from multiple search engines.
* Organize and share MS/MS experimental data with clients or colleagues."
"SEQUEST correlates uninterpreted tandem mass spectra of peptides with amino acid sequences from protein and nucleotide databases. SEQUEST will determine the amino acid sequence and thus the protein(s) and organism(s) that correspond to the mass spectrum being analyzed."