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QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification

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Abstract

Systems biology requires knowledge of the absolute amounts of proteins in order to model biological processes and simulate the effects of changes in specific model parameters. Quantification concatamers (QconCATs) are established as a method to provide multiplexed absolute peptide standards for a set of target proteins in isotope dilution standard experiments. Two or more quantotypic peptides representing each of the target proteins are concatenated into a designer gene that is metabolically labelled with stable isotopes in Escherichia coli or other cellular or cell-free systems. Co-digestion of a known amount of QconCAT with the target proteins generates a set of labelled reference peptide standards for the unlabelled analyte counterparts, and by using an appropriate mass spectrometry platform, comparison of the intensities of the peptide ratios delivers absolute quantification of the encoded peptides and in turn the target proteins for which they are surrogates. In this review, we discuss the criteria and difficulties associated with surrogate peptide selection and provide examples in the design of QconCATs for quantification of the proteins of the nuclear factor κB pathway.

Workflow for QconCAT mediated protein quatification

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Abbreviations

Glufib:

Glufibrinopeptide B

GPMDB:

Global Proteome Machine Database

GST:

Glutathione S-transferase

His-tag:

Hexahistidine purification tag

MALDI-ToF:

Matrix-assisted laser desorption/ionisation time of flight

MRM:

Multiple reaction monitoring

MS:

Mass spectrometry

NF-κB:

Nuclear factor κB

PrEST:

Protein epitope signature tag

PSAQ:

Protein standards for absolute quantification

QconCAT:

Quantification concatamer

Rel:

Reticuloendotheliosis

SDS-PAGE:

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

SILAC:

Stable isotope labelling by amino acids in cell culture

SRM:

Selected reaction monitoring

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Acknowledgments

D.M.S. and R.J.B. gratefully acknowledge funding from the Biotechnology and Biological Sciences Research Council (BBSRC) Systems Approach to Biological Research (SABR) grant BB/F005938/1 and BBSRC LOLA grant BB/G009112/1.

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Authors and Affiliations

  1. Protein Function Group, Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK

    Deborah M. Simpson & Robert J. Beynon

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  1. Deborah M. Simpson
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  2. Robert J. Beynon
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Correspondence to Robert J. Beynon.

Additional information

Published in the topical issue Quantitative Mass Spectrometry in Proteomics with guest editors Bernhard Küster and Marcus Bantscheff.

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Simpson, D.M., Beynon, R.J. QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification. Anal Bioanal Chem 404, 977–989 (2012). https://doi.org/10.1007/s00216-012-6230-1

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  • DOI: https://doi.org/10.1007/s00216-012-6230-1

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