Quantitative proteomics is a major focus of research activities at the Proteomics Centre with several different externally funded research projects based on bottom-up proteomic approaches (peptides surrogating proteins) in unbiased and targeted manners. For unbiased quantitative proteome analysis, the Centre’s research is based on the iTRAQ technology and other labeling techniques for relative protein quantitation. A key focus is the development and validation of procedures and protocols including standard operating protocols (SOPs) allowing the Centre to achieve optimal analytical performance and high reproducibility. All iTRAQ experiments are performed according to SOPs at the Proteomics Centre.
The Proteomics Centre’s research program in targeted protein quantitation is focused on the development of two complementary technologies capable of absolute protein quantitation, and their application to Biomarker Discovery and Validation in order to solve complex biomedical and environmental problems. These two technologies are: Multiple Reaction Monitoring, which is centered on a pure MS-based technique; and iMALDI, an approach that combines inexpensive and effective protein chip technology with the detection power of immuno Matrix-Assisted Laser Desorption/Ionization (iMALDI). While iMALDI is particularly suitable for quantitation of low-abundance proteins through the enrichment of target proteins by antibodies, MRM is advantageous due to its simplicity, speed, and ability to perform highly multiplex analysis (>1,000 proteins per analysis). Both MRM and iMALDI are valuable tools, saving time and expense, and will facilitate basic research involving large numbers of biological and environmental samples. Both technologies will provide information on environmental assessment and disease development, as well as identifying key targets for therapeutic and preventive drug development. In addition, MRM and iMALDI can be used in a clinical setting to provide "individualized medicine" -- for example, through detection of specific diagnostic peptides -- and can assist in both early detection of a patient's disease and selection of the most appropriate and most effective treatment.A recent review of current quantitative techniques in proteomics can be found here.
The Centre has developed and validated more than 40 SOPs for the iTRAQ procedure employing both LC-ESI-QTOF and LC-MALDI-TOF/TOF. Based on these SOPs coefficients of variation of less than 20% are achievable as demonstrated in a manuscript recently submitted for publication. The SOPs are currently extended for the use of the 8-plex iTRAQ reagents. Please visit Biomarker Discovery and Validation for additional information in iTRAQ.
The Centre is developing approaches in which iTRAQ is combined with specific enrichment strategies, including enrichment for phospho-peptides, to quantify changes in the modification level of proteins. Furthermore, the Centre develops approaches and techniques to improve the sensitivity of iTRAQ analysis. This includes specific sample handling steps to reduce the sample amount required and the implementation of exclusion lists in order to mine further down into the proteome. These exclusion lists are created from the first analytical round of data, which will be applied as a filter for re-analysis of the remaining sample. Previous experiments with the use of exclusion lists have resulted in the identification of approximately 50% more proteins than in the initial analyses.
- 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)
- Kuzyk MA, Ohlund LB, Elliott MH, Smith D, Qian H, Delaney A, Hunter CL, Borchers CH. 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).
- Lippert DN, Ralph SG, Phillips M, White R, Smith D, Hardie D, Gershenzon J, Ritland K, Borchers CH, Bohlmann J. Quantitative iTRAQ Proteome and Comparative Transcriptome Analysis of Elicitor-Induced Norway Spruce (Picea abies) Cells Reveals Elements of Calcium Signaling in the Early Conifer Defense Response. Proteomics. 9(2):350-67 (2009).
- Rosenzweig D, Smith D, Myler PJ, Olafson RW, Zilberstein D. Post-translational modification of cellular proteins during Leishmania donovani differentiation. Proteomics. 8(9):1843-50 (2008).
- Rosenzweig D, Smith D, Opperdoes F, Stern S, Olafson RW, Zilberstein D. Retooling Leishmania metabolism: from sand fly gut to human macrophage. Journal of the Federation of American Societies for Experimental Biology. 22(2): 590-602 (2008).
- Lucker J, Laszczak M, Smith D, Lund ST. Generation of a predicted protein database from EST data and application to iTRAQ analyses in grape (Vitis vinifera cv. Cabernet Sauvignon) berries at ripening initiation. BMC Genomics. 10:50. (2009). (published online 26 January 2009).
Immuno MALDI (iMALDI) derives from a mass spectrometric and immunoaffinity-based peptide chip technology developed in Dr. Borchers’ laboratory. Conventional sample preparation for MS protein analysis can be labor intensive and time consuming, especially if low-abundance proteins need to be detected and quantitated. However, iMALDI is a rapid method involving the use of antibodies to capture specific peptides and thus is well-suited for the quantitation of low-abundance proteins. These antibodies, which are bound to small beads for easy manipulation, can be deposited in rows on the surface of a small piece of glass or plastic to form a diagnostic "chip". The Centre offers iMALDI for fee-for-service or in collaboration through the development and implementation of specific iMALDI assays.
The Centre’s goals are to further develop, improve, multiplex, and automate the iMALDI technique; making iMALDI applicable for use in diagnostic clinics and research laboratories. By developing and improving reagents, protocols, robotic systems, and software, the entire iMALDI technology will be automated into a user-friendly diagnostics platform. All steps in the iMALDI process (i.e., sample preparation, chip fabrication, MS analysis, and data interpretation) will be integrated into a safe, efficient, and easy-to-use “candidate product” for the analysis of multiple proteins. For the development and commercialization of iMALDI, we have assembled a team from academia, clinical, environmental and research organizations, and industry, providing expertise in clinical diagnostics, ecology, environmental assessment, mass spectrometry, protein chemistry, immunology, robotics, engineering, software development and business. The ultimate goal of this interdisciplinary team is to provide the community with a robust, cost-effective, safe, and easy-to-use diagnostic device for absolute quantitation of the expression and modification levels of multiple proteins in a rapid and simultaneous manner, with the accuracy and specificity required for clinical, biological, and ecological samples.
- Reid, J.D., Holmes, D.T., Mason, D.R., Shah, B., Borchers, C.H. Towards the Development of an Immuno MALDI (iMALDI) Mass Spectrometry Assay for the Diagnosis of Hypertension JASMS, (2010)
- Jiang, J., Parker, C.E., Fuller, J.R., Kawula, T.H., Borchers C.H., An immunoaffinity tandem mass spectrometry (iMALDI) assay for detection of Francisella tularensis. Analytica Chimica Acta. 605: 70 -79 (2007).
- 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).
- Reid, J.D., Parker, C.E., Borchers C.H., Protein arrays for biomarker discovery. Current Opinion in Molecular Therapeutics. 9: 222-230 (2007).
- 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).
- 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).
- Warren, E.N., Elms, P.J., Parker, C.E, Borchers C.H., Development of a Protein Chip: A MS-based Method for Quantitation of Protein Expression and Modification Levels using an Immunoaffinity Approach. Analytical Chemistry. 76: 4082-4092 (2004).
Multiple Reaction Monitoring (MRM) is capable of accurate quantitation of numerous proteins in complex systems within a single experiment and is therefore ideal for biomarker validation and biomarker discovery. Although (MRM) assays have been widely employed for many years in pharmaceutical companies to quantify small molecules in biological specimens, its applicability towards quantitation of peptides for e.g. in blood samples has just been demonstrated recently. In particular, the Centre’s research is focused on the development of MRM approaches that use isotopically-coded peptides as standards enabling absolute quantitation of multiple proteins. As part of the Centre’s research program, cocktails of these standards are currently being created for profiling hundreds of proteins including blood proteins linked to cardiovascular diseases (CVD); for examining the endocrine pathways which can be disrupted by environmental perturbation; and for quantifying enzymes involved in the defense mechanism of trees against microbial attack. The specific proteins that we are targeting in the project of elucidating the defense mechanism are functionally different but structurally very similar and therefore difficult to quantify by the more-common ELISA technique. The peptide cocktails developed for the CVD project will be employed in large-scale proteomic analyses. As can be seen from these few examples, these “cocktails” have great potential for a wide range of applications.
Standard peptides are available through the Proteomics Centre Peptide Synthesis Services. Peptide cocktails and MRM methods currently developed are available by contacting the Centre directly. Read more on our ever-expanding list of MRM assays for human and mouse plasma proteins by clicking here. Furthermore, the Centre offers the development and implementation of complete and customized MRM assays as well as conducting these assays.
- Kuzyk MA, Smith D, Yang J, Cross T, Jackson A, Hardie D, , Anderson NL, Borchers CH. MRM-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma. Molecular and Cellular Proteomics, Published online (May 2009).