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

Nature Protocols volume 1, pages 1029–1043 (2006)Cite this article

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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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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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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