Biomarker Discovery and Validation

The UVic Genome BC Proteomics Centre offers protein expression analysis services for biomarker discovery and validation. Differential labeling technologies, such as iTRAQ and SILAC, are ideal for determining relative changes in protein expression in sets of samples leading to the discovery of potential biomarkers. These technologies are further complemented by comprehensive approaches to establish validity of specific proteins as biomarkers. Comprehensive data includes absolute quantitation of targeted proteins and post-translational modifications. The Centre offers several methods of absolute quantitation, including Multiple Reaction Monitoring (MRM) and immuno-MALDI (iMALDI) an approach combining peptide enrichment by anti-peptide antibodies and MS by direct MALDI-MS and MS/MS analysis of immunoprecipitated peptides (1, 2, 3).

Biomarker Discovery

The iTRAQ technology is ideal for comparing normal, diseased, and drug-treated samples, time course studies, biological replicates and relative quantitation. Applied Biosystems iTRAQ isobaric affinity labels allow for multiplexing up to 4 or 8 samples per experiment. After tryptic digestion, multiple peptides are labeled, including peptides with post translational modifications. Our approach includes strong cation exchange prior to LC-MS/MS analysis on our ABI QSTAR Pulsar i or LC-MALDI TOF/TOF analysis on our ABI 4800 MALDI TOF/TOF (4, 5, 6). Standard Operating Protocols and Quality Control Procedures developed by the Proteomics Centre minimize analytical variability in iTRAQ. Please refer to the Applied Biosystems website for further information on iTRAQ.

Advantages to the iTRAQ technology include:
  • Ideal for comparative studies as up to 4 or 8 labels can be used for multiplexing experiments
  • Non-targeted approach for discovery studies
  • Improved MS/MS fragmentation results in higher confidence protein identifications
  • Post translational modifications can be detected
  • Relative quantitation
  • SOP-driven work flow
  • Established Quality Control procedures

Biomarker Validation

Absolute quantitative data, including protein concentration, post-translational modification status, and copy number/cell is important for biomarker studies. In addition to validating potential biomarkers, absolute quantitation allows for simplified bioinformatics analysis and comparison of numerous samples. At the UVic Genome BC Proteomics Centre, two different approaches allow for absolute quantitation of low and moderate to highly abundant proteins. MRM is a technique that uses multiple stages of mass spectrometry to achieve high levels of sensitivity and specificity in the quantitation of proteins from a complex mixture. Similarly to iTRAQ analysis, proteolytic peptides are separated by chromatography prior to mass spectrometry analysis on a triple quadrupole instrument (QTRAP 2000 or QTRAP 4000). Specificity is achieved through the precise selection of a targeted peptide mass in the first quadrupole and a specific fragment ion mass in the third quadrupole. Absolute quantitation is achieved through the addition of isotopically-coded internal standards. The Proteomics Centre offers custom peptide synthesis as a service, including isotopically coded peptides for absolute quantitation. MRM is ideal for absolute quantitation of medium to highly abundant proteins. Please see the poster "MRM based, multiplexed, absolute quantitation of 32 high abundance proteins in human plasma" for further information.

Our immuno-MALDI (iMALDI) peptide chip technology is another comprehensive method of protein expression analysis which we are currently developing for multiplex analysis (7, 8). Proteins of interest are proteolytically cleaved and the resulting peptides are captured using anti-peptide antibodies immobilized on affinity beads. The affinity-bound peptides are analyzed by MALDI TOF mass spectrometry, and tandem MS (MS/MS) for specificity. Like the MRM technology, isotopically-coded internal standards are incorporated for absolute quantitation. The specificity and sensitivity of iMALDI is well suited to the analysis of low abundant proteins.

Further experimental details for these applications can be found in these relevant publications:
  1. Cohen Freue, G.V., Sasaki, M., Meredith, A., Gunther, O.P., Bergman, A., Takhar, M., Mui, A., Balshaw, R.F., Ng, R.T., Opushneva, N., Hollander, Z., Li, G., Borchers, C., Wilson-McManus, J., McManus, B.M., Keown, P.A., McMaster, W.R. Proteomic signatures in plasma during early acute renal allograft rejection Mol Cell Proteomics, 9: 1954-1967. (2010)
  2. Reid, J.D., Parker, C.E. Borchers, C.H. Protein arrays for biomarker discovery Current Opinion in Molecular Therapeutics, 9(3):216-21 (2007)
  3. Jiang, J., Parker, C.E., Robinette, D., Borchers, C.H. A Clinical Diagnostics Platform - Protein Detection Using an Immunoaffinity-based Peptide Chip BIOforum Europe, 10/2006: 18-20 (2006)
  4. Warren, E.N., Jiang, J., Parker, C.E., Borchers C.H., Absolute Quantitation of Cancer-related Proteins using an MS-based Peptide Chip. BioTechniques, 38: S7-S11 (2005)
  5. Rosenzweig, D., Smith, D., Myler, P. J., Olafson, R.W., Zilberstein, D. Post-translational modification of cellular proteins during Leishmania donovani differentiation Proteomics, 8:1843-1850 (2008)
  6. Rosenzweig, D., Smith, D., Opperdoes, F., Stern, S., Olafson, R.W., Zilberstein, D. Retooling Leishmania metabolism: from sand fly gut to human macrophage. FASEB Journal, 22(2):590-602 (2008)
  7. Kuzyk, M.A., Ohlund, L.B., Elliott, M.H., Smith, D., Qian, H., Delaney, A., Hunter, C.L., Borchers, C.H. A comparison of MS/MS-based, stable-isotope-labeled, quantitation performance on ESI-QqTOF and MALDI-TOF/TOF mass spectrometers. Proteomics, 5;9(12):3328-3340 (2009).
  8. Jiang, J., Parker, C.E., Fuller, J.R., Kawula, T.H., Borchers, C.H., Wainwright C.E., Wainwright B.J. An Immunoaffinity Tandem Mass Spectrometry (iMALDI) assay for Detection of Francisella tularensis. Analytica Chimica Acta, Dec 12; 605(1):70-9. Epub 2007 Oct 23. PMID: 18022413 (2007)
  9. Jiang, J., Parker C.E., Hoadley, K.A., Perou, C.M., Borchers C.H., Development of an Immuno Tandem Mass Spectrometry (iMALDI) Assay for EGFR Diagnosis, Proteomics Clin. Appl. 1, 1651-1659 (2007)