Easy access for the scientific community

The past decade has witnessed an explosion in the field of proteomics. This development has been driven by the development of database search algorithms, expansion of sequence databases and improvements in mass spectrometry instrumentation. Quantitative techniques using isotopic dilution have allowed quantitative experiments. The expanding opportunities have propelled the development of core facilities to provide services to biological researchers wishing to conduct proteomic experiments for themselves.

The high-throughput proteomics facility at the Institute for Systems Biology (Seattle, WA, USA) is a state-of-the-art resource involved in all aspects of proteomic research. Work includes, but is not limited to, sample preparation, chromatographic purification, analysis on a variety of mass spectrometers, database search using SEQUEST1 and validation and interpretation of the results for our clients.

Samples typically come from other research groups within our institute or local collaborators, though it is not infrequent for samples to originate in other parts of the country or world. The turnaround time for sample analysis is approximately one week, depending on the type of processing required and the size of the queue. A Web site www.systemsbiology.proteomicsqueue.org provides information on instrument status, queue size and the status of individual projects. The facility has a full time staff of three operators, each of whom specialises in the operation of one type of spectrometer, but also provides support to the rest of the staff as needed.

The proteomics facility offers sample preparation options that include highly sophisticated labeling techniques such as ICAT (Isotope Coded Affinity Tag) and iTRAQ, available from Applied Biosystems (Foster City, CA). Both ICAT and iTRAQ labels are used to provide relative abundances of samples to be compared, i.e. wild-type and abnormal phenotypes, which are then analysed in a single, combined sample. Because the facility was created as adjunct to the Aebersold lab, where the ICAT reagent was developed and tested2,3, we have extensive experience in the preparation with this technique. ICAT chemistry uses two labels that differ in mass through the incorporation of carbon 13 into one of the pair. The isotope coded affinity tag, consisting of a thiol reactive isotope coded tag bound through an acid-labile linker to a biotin molecule, is used to covalently modify the side chain of cysteine residues in proteins. Following enzymatic digestion, cysteine containing labelled peptides are captured using an avidin derivitized resin. The biotin is removed before MS analysis through cleavage in strong acid. This reagent has had significant success in quantifying changes in biological systems4,5. In collaboration with the proteomics facility, Ranish et al. utilised the ICAT labeling to identify TFB5, a new component of general transcription and DNA repair factor IIH6.

The facility also has substantial hands-on experience using the iTRAQ reagent to perform quantitative proteomics experiments. The iTRAQ reagent employs four reporter ions of masses 114, 115, 116 and 117 Da that are created during collisional dissociation. These labels are covalently bound to the N-terminus of each polypeptide and the amine side chain of lysine residues. During the MS/MS analysis the isotopic labels to fragment from the peptide, producing signature peaks of the label. Therefore, peptide assignment and multiplex quantification are determined from the same spectrum. Four samples can be compared using this reagent by comparing the intensity of signature peaks from each of the labels. Since the signature peaks are small (114-117 Da), it is necessary to have a mass spectrometer capable of measuring in this low mass region. Our facility uses hybrid quadrupole time of flight instruments for this purpose.

The protoemics facility is equipped with multiple HPLC instruments and routinely employs offline separation as needed to separate complex mixtures to concentrations appropriate for analysis by mass spectrometry. The main chromatographic separations utilised are strong cation exchange with polysulfoethyl A columns from PolyLC (Columbia, MD). An ABI Vision workstation allows high throughput preparation of ICAT samples by combining cation exchange separation and avidin affinity chromatography in one instrument.

The heart of the proteomics facility is the mass spectrometers. Five mass spectrometers are available for analysis of peptide samples. These include two ion traps, two quadrupole time of flight (TOF) instruments and a MALDI-TOF-TOF instrument. This combination has been selected to provide accurate data for a range of applications, including optimisation for each of the isotope labels.

Each instrument is automated allowing for near continuous operation7. The automation instrument employs a Famos autosampler (Dionex, Sunnyvale, CA) to load samples onto a 1cm x 100 μm i.d. pre-column of Magic 5μ C18aq (Michrom Bioresources, Auburn, CA) with 200 angstrom pore. After washing a gradient is run eluting peptides onto a 75mm i.d. fused silica capillary packed in-house with 10cm of Magic 5μ C18aq with 100 angstrom pore. Gradients are typically run for 60 to 120 minutes with flow rates of 200 nl/min. All LC-MS instruments are equipped with an in-house built microspray device.

The ion trap mass spectrometers in the facility consist of a Thermo (San Jose, CA) LCQ Deca and LTQ. These instruments provide excellent sensitivity with a high sampling frequency that permits a large number of peptide identifications even in complex samples. The instruments are operated using the dynamic exclusion option with multiple data dependent scans acquired for each precursor scan. These instruments are well suited for quantifying samples labeled with ICAT chemistry as well as individual samples such as immunoprecipitations or in-gel digests where high sensitivity is required.

The facility also operates two hybrid quadrupole-TOF instruments, a Waters (Milford, MA) Ultima QTOF and an Applied Biosystems API QSTAR Pulsar i. Time of flight instruments provide a high level of mass accuracy and resolution for both TOF-MS and TOF-MS/MS analysis. In addition, quadrupole-TOF mass spectrometers are capable of detecting low molecular weight molecules, essential for analysing samples labeled with iTRAQ. These instruments also have advanced information dependent acquisition capabilities, allowing peptides to be selected for CID based on charge state, abundance, mass and other parameters. Furthermore, the collision energy during CID can be optimised for peptide mass and charge state. These features enhance the quality of the data acquired on these instruments.

Finally, the facility has available an ABI 4700 TOF/TOF instrument. This is used for the rapid analysis of samples prepared by in-gel digest as well as method development.

Sample handling in the proteomics facility also includes database searching and subsequent analysis using a suite of software developed at the Institute for Systems Biology. Data from all instruments is converted to the mzXML format8 and searched using Sequest. All search results are then filtered for probability of correct assignment using PeptideProphet9. Each peptide is then assigned to a protein using a second program called ProteinProphet10. Each protein assignment is given a probability based on the combined probability of each of the matched peptides. The results are a protein dataset that is both accurate and predictable, significantly reducing the time spent on manual validation of spectra. All proteins are hyperlinked through the source database to allow the user to rapidly assess experimental results. Additional algorithms provide quantification of isotopic masses, allowing the ICAT and iTRAQ labeling techniques to be fully utilised11. All software analysis tools are available for download from the Institute for Systems Biology at http://cvs.sourceforge.net/ viewcvs.py/sashimi/trans_proteomic_pipeline/.

It is our goal to provide easy access to mass spectrometry for the scientific community both locally and globally. Our services and expertise cover most aspects of sample preparation, mass spectrometry and data analysis. A wide variety of instrumentation is available to accommodate most research needs.

References

1. Eng, J.; McCormack, A.; Yates III, J. R. J. Am. Soc. Mass Spectrom 1994, 976-989.
2. Yi, E. C.; Li, X. J.; Cooke, K.; Lee, H.; Raught, B.; Page, A.; Aneliunas, V.; Hieter, P.; Goodlett, D. R.; Aebersold, R. Proteomics 2005, 5, 380-387.
3. Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat Biotechnol 1999, 17, 994-999.
4. Martin, D. B.; Gifford, D. R.; Wright, M. E.; Keller, A.; Yi, E.; Goodlett, D. R.; Aebersold, R.; Nelson, P. S. Cancer Res 2004, 64, 347-355.
5. Yan, W.; Lee, H.; Deutsch, E. W.; Lazaro, C. A.; Tang, W.; Chen, E.; Fausto, N.; Katze, M. G.; Aebersold, R. Mol Cell Proteomics 2004, 3, 1039-1041.
6. Ranish, J. A.; Hahn, S.; Lu, Y.; Yi, E. C.; Li, X. J.; Eng, J.; Aebersold, R. Nat Genet 2004, 36, 707-713.
7. Yi, E. C.; Lee, H.; Aebersold, R.; Goodlett, D. R. Rapid Commun Mass Spectrom 2003, 17, 2093-2098.
8. Pedrioli, P. G. A.; Eng, J. K.; Hubley, R.; Vogelzang, M.; Deutsch, E. W.; Raught, B.; Pratt, B.; Nilsson, E.; Angeletti, R. H.; Apweiler, R.; Cheung, K.; Costello, C. E.; Hermjakob, H.; Huang, S.; Julian, R. K.; Kapp, E.; McComb, M. E.; Oliver, S. G.; Omenn, G.; Paton, N. W.; Simpson, R.; Smith, R.; Taylor, C. F.; Zhu, W.; Aebersold, R. 2004, 22, 1459-1466.
9. Keller, A.; Nesvizhskii, A. I.; Kolker, E.; Aebersold, R. Anal Chem 2002, 74, 5383-5392.
10. Nesvizhskii, A. I.; Keller, A.; Kolker, E.; Aebersold, R. Anal Chem 2003, 75, 4646-4658.
11. Li, X. J.; Zhang, H.; Ranish, J. A.; Aebersold, R. Anal Chem 2003, 75, 6648-6657.