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Targeted quantitative proteomics

Whereas shotgun proteomics provides global, untargeted proteome inventories with quantitative estimates, many studies require more precise, targeted measurements of proteins.  We employ liquid chromatography-multiple reaction monitoring (MRM) mass spectrometry to do targeted measurements of individual proteins.  In this approach, we first select one or more peptide sequences unique to the target protein.  We then use a triple quadrupole MS instrument to detect specific MRM transitions that represent each peptide sequence.  This analysis selectively detects the target peptides, even in complex mixtures, such as tryptic digests from cells, tissues and biofluids.  Precise quantitation is achieved by incorporating stable isotope labeled standards for each peptide (stable isotope dilution) or by using a labeled peptide standard for multiple sequences.  This approach offers a method to systematically configure assays for individual proteins, without the requirement for antibodies.  A key advantage of MRM is that analyses may be multiplexed, such that peptides representing multiple proteins can be analyzed in the same LC-MS analysis.  This permits development of MRM assay panels built around biological themes (groupings of related proteins) or groups of candidate protein biomarkers.   A key enabling resource for MRM assay design and data analysis is Skyline, which was developed by the MacCoss laboratory with support from the Vanderbilt CPTAC program.

 

Representative references from our work and collaborations

Addona, T. A., Abbatiello, S. E., Schilling, B., Skates, S. J., Mani, D. R., Bunk, D. M., Spiegelman, C. H., Zimmerman, L. J., Ham, A. J., Keshishian, H., Hall, S. C., Allen, S., Blackman, R. K., Borchers, C. H., Buck, C., Cardasis, H. L., Cusack, M. P., Dodder, N. G., Gibson, B. W., Held, J. M., Hiltke, T., Jackson, A., Johansen, E. B., Kinsinger, C. R., Li, J., Mesri, M., Neubert, T. A., Niles, R. K., Pulsipher, T. C., Ransohoff, D., Rodriguez, H., Rudnick, P. A., Smith, D., Tabb, D. L., Tegeler, T. J., Variyath, A. M., Vega-Montoto, L. J., Wahlander, A., Waldemarson, S., Wang, M., Whiteaker, J. R., Zhao, L., Anderson, N. L., Fisher, S. J., Liebler, D. C., Paulovich, A. G., Regnier, F. E., Tempst, P., and Carr, S. A. (2009) Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Nat Biotechnol, 27, 633-641.  PubMed

Zhang, H., Liu, Q., Zimmerman, L. J., Ham, A. J., Slebos, R. J., Rahman, J., Kikuchi, T., Massion, P. P., Carbone, D. P., Billheimer, D., and Liebler, D. C. (2011) Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry. Mol Cell Proteomics, 10, M110 006593.  PubMed

MacLean, B., Tomazela, D. M., Shulman, N., Chambers, M., Finney, G. L., Frewen, B., Kern, R., Tabb, D. L., Liebler, D. C., and MacCoss, M. J. (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics, 26, 966-968.  PubMed