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Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes

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Abstract

An important area of proteomics involves the need for quantification, whether relative or absolute. Many methods now exist for relative quantification, but to support biomarker proteomics and systems biology, absolute quantification rather than relative quantification is required. Absolute quantification usually involves the concomitant mass spectrometric determination of signature proteotypic peptides and stable isotope-labeled analogs. However, the availability of standard labeled signature peptides in accurately known amounts is a limitation to the widespread adoption of this approach. We describe the design and synthesis of artificial QconCAT proteins that are concatamers of tryptic peptides for several proteins. This protocol details the methods for the design, expression, labeling, purification, characterization and use of the QconCATs in the absolute quantification of complex protein mixtures. The total time required to complete this protocol (from the receipt of the QconCAT expression plasmid to the absolute quantification of the set of proteins encoded by the QconCAT protein in an analyte sample) is ∼29 d.

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Figure 1
Figure 2: Overall workflow for QconCAT quantification.
Figure 3
Figure 4: Induction of QconCAT protein in BL21(λDE3) cells grown in Luria broth.
Figure 5: HisTrap purification of QconCAT.
Figure 6: ESI-MS to measure the intact mass of QconCATs.
Figure 7: Incorporation of label into peptides derived from QconCATs.
Figure 8: Quantification by QconCATs.
Figure 9: Quantification of biological samples by QconCAT.

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References

  1. Julka, S. & Regnier, F.E. Recent advancements in differential proteomics based on stable isotope coding. Brief. Funct. Genomic. Proteomic. 4, 158–177 (2005).

    Article  CAS  Google Scholar 

  2. Ong, S.E. & Mann, M. Mass spectrometry–based proteomics turns quantitative. Nat. Chem. Biol. 1, 252–262 (2005).

    Article  CAS  Google Scholar 

  3. Yan, W. & Chen, S.S. Mass spectrometry–based quantitative proteomic profiling. Brief. Funct. Genomic. Proteomic. 4, 27–38 (2005).

    Article  CAS  Google Scholar 

  4. Gerber, S.A., Rush, J., Stemman, O., Kirschner, M.W. & Gygi, S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. USA 100, 6940–6945 (2003).

    Article  CAS  Google Scholar 

  5. Kirkpatrick, D.S., Gerber, S.A. & Gygi, S.P. The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications. Methods 35, 265–273 (2005).

    Article  CAS  Google Scholar 

  6. Beynon, R.J., Doherty, M.K., Pratt, J.M. & Gaskell, S.J. Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides. Nat. Methods 2, 587–589 (2005).

    Article  CAS  Google Scholar 

  7. Wu, C.C. & Yates, J.R. The application of mass spectrometry to membrane proteomics. Nat. Biotechnol. 21, 262–267 (2003).

    Article  CAS  Google Scholar 

  8. Lu, X. & Zhu, H. A novel proteomic approach for high throughput analysis of membrane proteins. Mol. Cell Proteomics 4, 1948–1958 (2005).

    Article  CAS  Google Scholar 

  9. Fischer, F. & Poetsch, A. Protein cleavage strategies for an improved analysis of the membrane proteome. Proteome Sci. 4, 1–12 (2006).

    Article  Google Scholar 

  10. Brancia, F.L. et al. A combination of chemical derivatisation and improved bioinformatic tools optimises protein identification for proteomics. Electrophoresis 22, 552–559 (2001).

    Article  CAS  Google Scholar 

  11. Beardsley, R.L. & Reilly, J.P. Optimization of guanidination procedures for MALDI mass mapping. Anal. Chem. 74, 1884–1890 (2002).

    Article  CAS  Google Scholar 

  12. Thevis, M., Ogorzalek Loo, R.R. & Loo, J.A. In-gel derivatization of proteins for cysteine-specific cleavages and their analysis by mass spectrometry. J. Proteome Res. 2, 163–172 (2003).

    Article  CAS  Google Scholar 

  13. Beynon, R.J. & Pratt, J.M. Metabolic labelling of proteins for proteomics. Mol. Cell Proteomics 4, 857–8872 (2005).

    Article  CAS  Google Scholar 

  14. Ellison, D., Hinton, J., Hubbard, S.J. & Beynon, R.J. Limited proteolysis of native proteins: the interaction between avidin and proteinase K. Protein Sci. 4, 1337–1345 (1995).

    Article  CAS  Google Scholar 

  15. Hubbard, S.J. The structural aspects of limited proteolysis of native proteins. Biochim. Biophys. Acta 1382, 191–206 (1998).

    Article  CAS  Google Scholar 

  16. Hubbard, S.J., Beynon, R.J. & Thornton, J.M. Assessment of conformational parameters as predictors of limited proteolytic sites in native protein structures. Protein Eng. 11, 349–359 (1998).

    Article  CAS  Google Scholar 

  17. Wu, C., Robertson, D.H., Hubbard, S.J., Gaskell, S.J. & Beynon, R.J. Proteolysis of native proteins. Trapping of a reaction intermediate. J. Biol. Chem. 274, 1108–1115 (1999).

    Article  CAS  Google Scholar 

  18. Hubbard, S.J. & Beynon, R.J. Proteolysis of native proteins as a structural probe. In Proteolytic Enzymes. A Practical Approach (eds. Beynon, R.J. & Bond, J.S.) 233–264 (Oxford University Press, Oxford, 2001).

    Google Scholar 

  19. Sambrook, J. & Russell, D.W. (eds.) Preparation and transformation of competent E. coli using calcium chloride (Protocol 25). In Molecular Cloning: A Laboratory Manual 3rd Edn. Vol 1. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

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Acknowledgements

This work was supported by the Biotechnology and Biological Sciences Research Council, Swindon, UK.

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Authors and Affiliations

  1. Department of Veterinary Preclinical Sciences, University of Liverpool, Liverpool, L69 7ZJ, Crown Street, UK

    Julie M Pratt, Deborah M Simpson, Mary K Doherty, Jenny Rivers & Robert J Beynon

  2. Michael Barber Centre for Mass Spectrometry, School of Chemistry, University of Manchester, Manchester, M13 9PL, UK

    Simon J Gaskell

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  1. Julie M Pratt
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  2. Deborah M Simpson
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  3. Mary K Doherty
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  4. Jenny Rivers
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  5. Simon J Gaskell
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  6. Robert J Beynon
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Corresponding author

Correspondence to Robert J Beynon.

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Competing interests

JMP, RJB and SJG are the inventors of the QconCAT technology, and the license to create QconCAT proteins resides exclusively with Entelechon GmBH.

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Pratt, J., Simpson, D., Doherty, M. et al. Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes. Nat Protoc 1, 1029–1043 (2006). https://doi.org/10.1038/nprot.2006.129

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