We use a bottom-up proteomics scheme to identify proteins. We carry out protein identification experiments on a variety of sample types, ranging from a single band observed on an SDS-PAGE gel to highly complex protein mixtures isolated from cellular lysates.
In our process, we digest protein samples with a protease, typically trypsin, and subject the proteolytic peptides to tandem mass spectrometry, or MS/MS. We then compare the resulting MS/MS data to protein databases via automated protein database searching to identify the proteins that contain the peptides detected by the mass spectrometer. Sample scales can range from sub-microgram up to hundreds of micrograms.
We perform a number of common quantitative analyses, including label-free quantification (area under the curve), spectral counting, stable isotope labeling by amino acids in cell culture (SILAC), isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT). Each quantification technique has its own strengths, limitations and requirements. We can consult with you to help you determine the appropriate analysis for your sample type.
Phosphorylation, acetylation, methylation and ubiquitylation are common modifications we analyze, but analysis of other modifications is possible. Analyses we conduct range from peptide mapping experiments on a single protein to multiplexed, proteome-wide modification studies. We are especially experienced with qualitative and quantitative analysis of modifications on histones. Since there are over 300 known protein modifications, we advise investigators to consult with us during the design phase of the experiments in order to develop the appropriate strategy for mass spectrometric analysis of the modification.
We provide expertise in preparing samples, primarily proteins and peptides for mass spectrometric analysis. Our sample preparation services include enzymatic digestion of proteins in solution or in gel slices, and sample desalting and concentration. We also have extensive expertise in preparing samples, such as from serum or cell lysates, for multiplex quantitative analyses using iTRAQ and TMT isobaric chemical tags. High-performance liquid chromatography, or HPLC, purification is available for the purification and/or fractionation of protein and peptide mixtures via ion-exchange and reverse/basic-reverse phase separations. We also offer isolation and enrichment of phosphopeptides via metal-based affinity techniques. We encourage investigators to consult with us on relevant aspects of experimental design, such as estimating protein quantities needed for analysis and identifying contaminants that might inhibit analysis.
We maintain the following HPLC instrumentation:
Each HPLC is equipped with a fraction collector that can collect into a variety of formats, including deep or regular well 96-well plates and 15 mL tubes. The Dynamax HPLC is ideal for large-scale purification of synthetic peptides and peptide conjugation products. The Vanquish and Paradigm instruments are frequently used for basic reverse-phase fractionation of complex peptide mixtures that will be subsequently analyzed by mass spectrometry and ion-exchange separations of globin chains for hemoglobin variant analysis. Contact us to inquire about the use of these instruments.
We maintain a Thermo Scientific Proteome Discoverer v2.4 (PD 2.4) for analyzing data from virtually all liquid chromatography electrospray ionization, or LC-ESI, data, including data from protein identification, quantification (including label-free, spectral counting, SILAC, TMT and iTRAQ data), and protein modification experiments. PD2.4 is operated on an OmicsPCs Maximum Destroyer Ultra desktop computer equipped with 36 processors/72 threads for high-speed data analysis. We also use the open-source software Skyline for the analysis of data from parallel reaction monitoring and selective reactive monitoring, or PRM and SRM. Investigators can receive assistance with downstream data analysis (e.g., significance testing, pathway analysis, protein interaction analysis) from us as well as from the bioinformatics team in the Genomics & Bioinformatics shared resource.
Users can view their results in either Microsoft Excel outputs or the Proteome Discoverer viewer, which will allow for filtering, sorting and downstream processing of the results. Please contact us for details about data processing, because we must construct experiment-specific workflows.
To download the Proteome Discoverer viewer, visit the software download and licensing portal and create an account. Then, download Proteome Discoverer and update existing software if needed (be sure to follow instructions on prerequisite software required to run Proteome Discoverer). A fully functional, trial version of Proteome Discoverer will be installed on your computer. After 30 days the trial version will only act as a viewer of output files. Proteome Discoverer is compatible with computers on Windows-based operating systems.
We periodically offer a week-long course on mass spectrometry-based proteomics taught by core Director Dr. Phil Gafken. The course is recommended for those who currently use proteomics or who plan to use proteomics in the near future. The course is intended to provide novice users with a solid understanding of the technology they could apply to their research.
The course covers the following topics:
The class is free to Fred Hutch employees. Space is usually limited. Contact Dr. Phil Gafken to learn more.
Other training resources
The Thermo Scientific Orbitrap Eclipse combines quadrupole, OrbiTrap and ion trap mass analyzers into a single instrument, called a Tribrid design, that provides high data acquisition flexibility and utility. The Orbitrap analyzer can obtain data at resolutions ranging from 7,500 to 500,000 (at m/z 200) with mass accuracy of <3 ppm. The unique design and data acquisition strategies allow the Eclipse to acquire data at 45 Hz with the ion trap and 40 Hz with the Orbitrap. A field asymmetric ion mobility spectrometry, or FAIMS, unit is also mounted on the Eclipse. This provides gas-phase ion mobility separations to increase the signal to noise ratio of ions entering the mass spectrometer and to increase overall data quality.
Like the older generation Fusion, the Eclipse is equipped with synchronous precursor selection, or SPS, for selecting MS2 precursors for MS3 analysis to decrease the effects of precursor co-isolation during MS1, resulting in increased quantification accuracy for TMT and iTRAQ experiments. The Eclipse is also equipped with real-time database searching to increase the utility and success rate of MS3 quantification scans. A Thermo Scientific Easy-nLC 1200 UHPLC is coupled to the Fusion to provide liquid chromatography mass spectrometry, or LC-MS, at flow rates of 200 nL/min. to 800 nL/min. and at either conventional or ultrahigh-pressure liquid chromatography conditions.
Recommended uses: This system is primarily used for protein identification and quantification (SILAC, TMT, iTRAQ, label-free, spectral counting) of samples with very high complexity (e.g., lysates) and for global protein modification characterization (phosphoproteomics).
The Thermo Scientific Orbitrap Fusion combines quadrupole, OrbiTrap and ion trap mass analyzers into a single instrument, called a Tribrid design, that provides high data acquisition flexibility and utility. The Orbitrap analyzer can obtain data at resolutions ranging from 15,000 to 450,000 (at m/z 200) with mass accuracy of <1 ppm. The unique design and data acquisition strategies allow the Fusion to acquire data at 20 Hz with the ion trap and 15 Hz with the Orbitrap.
The Fusion is equipped with SPS for selecting MS2 precursors for MS3 analysis. This feature decreases the effects of precursor co-isolation during MS1 to increase quantification accuracy for TMT and iTRAQ experiments. The Fusion is also equipped with collision-induced dissociation, or CID, higher energy collision-induced dissociation, or HCD, and electron transfer dissociation, or ETD. A Thermo Scientific Easy-nLC 1000 UHPLC is coupled to the Fusion to provide LC-MS at flowrates of 200 nL/min. to 800 nL/min. and at either conventional or ultrahigh-pressure liquid chromatography conditions.
Recommended uses: This is primarily used for protein identification and quantification (SILAC, TMT, iTRAQ, label-free, spectral counting) of samples with very high complexity (e.g., lysates) and for global protein modification characterization (phosphoproteomics).
The Thermo Scientific Orbitrap Elite is a hybrid mass spectrometer containing both Orbitrap and ion trap mass analyzers. This instrument can achieve a mass resolution of 240,000 (at m/z 400), a mass accuracy <3 ppm, limits of detection <1 femtomole and tandem mass spectra recording speeds up to 8 Hz (ion trap). The Elite is equipped with CID, HCD and ETD, which provide complementary peptide fragmentation. The complementarity of these techniques has the potential to enhance peptide fragmentation for identification purposes and the characterization of post-translational modifications, especially labile modifications such as phosphorylation. HCD also supports the use of iTRAQ or TMT quantification approaches. The Elite is coupled to a Thermo Scientific Easy-nLC II for typical chromatography flowrates between 300 nL/min. to 600 nL/min.
Recommended uses: This system is used for protein identification and quantification (label-free, spectral counting, SILAC, TMT, iTRAQ) of samples with medium complexity (affinity purifications, bacterial lysates) and for protein modification characterization using peptide mapping.
The Thermo Scientific TSQ Vantage is a triple quadrupole mass spectrometer. The TSQ contains hyperbolic quadrupole rods for high ion transmission and it also contains an S-lens for thorough desolvation of ions, resulting in increased signal. This instrument uses an electrospray ionization source and it is capable of multiple scan functions, such as selective ion monitoring, or SIM, selective/multiple reaction monitoring, or S/MRM, and precursor ion scanning, which are not available on other instruments in our shared resource. This instrument is coupled to an Eksigent nano2D HPLC for performing LC-MS at flowrates ranging from 50 nL/min. to 50 μL/min.
Recommended uses: The primary use of this instrument is the qualitative and quantitative analysis of targeted peptides from complex mixtures and for quantifying small molecules.
The Applied Biosystems 4800 matrix-assisted laser-desorption ionization, or MALDI, mass spectrometer is equipped with tandem time-of-flight, or TOF-TOF, technology for performing tandem mass spectrometry (peptide sequencing). Mass resolutions of >20,000, mass accuracies of <20 ppm and limits of detection of <1 femtomole are possible for peptides. While the instrument can be quite useful for mass measurements of proteins, the mass resolution, mass accuracy and limits of detection degrade as molecular weight increases.
Recommended uses: The 4800 is ideal for supporting de novo sequencing of peptides, peptide mapping for detecting protein modifications, sample screening and mass measurements on peptides, proteins and oligonucleotides.