A variety of heavy isotope-labelled precursors are available for incorporation into QconCATs. The choice depends on budget, cleavage strategy and the amino acid distribution of the analyte.

QconCAT proteins can be made available in unlabelled and labelled forms for proteomic studies. The choice of label depends on the cleavage strategy, the amino acid distribution of proteins in the analyte and cost. PolyQuant produces labelled QconCATs by growing E. coli expressing the QconCAT in minimal medium plus amino acids, substituted with isotope labelled precursors of relative isotope abundance (RIA) >98%.

Heavy isotope-labelled precursors for labelling

PolyQuant labels the QconCAT proteins in a fully defined synthetic culture medium, containing isotope-labelled Arg and Lys residues – either 13C arg/lys (resulting in a mass difference of 6Da), or 13C15N arg/lys (resulting in a mass difference of 10Da/8Da) (1). In addition, full labelling of all nitrogen atoms is possible by using 15N ammonium chloride as the sole nitrogen source during expression. The latter labelling option, whilst relatively inexpensive, results in mass differences between labelled and unlabelled peptides being different for each peptide, depending on the number of nitrogen atoms. This must be taken into account when designing the QconCAT and can increase the complexity of data processing (2). Other labelling regimens are open for discussion (e.g. 13C leucine, valine, isoleucine or phenylalanine).

Labelling efficiency of expression in E. coli

The degree to which QconCAT proteins are labelled depends on the RIA of the precursor amino acids and the degree to which endogenous, rather than exogenous, amino acids are incorporated during protein synthesis. Endogenous amino acids can arise from de novo biosynthesis or protein turnover. Heavy isotope labelled amino acids and 15N labelled ammonium chloride are generally labelled to > 98%. This impacts the labelling efficiency of all heavy isotope labelled internal standards used in proteomics, whether derived by chemical synthesis, labelling in vivo or labelling in vitro. It is not currently possible to prepare any QconCAT (or indeed, any recombinant protein) that is 100% stable isotope labelled.

PolyQuant routinely achieves QconCAT labelling efficiencies equivalent to the starting RIA of the heavy isotope labelled precursor (i.e. ≥ 98%) (2,3). During expression, de novo amino acid biosynthesis pathways are suppressed because amino acids are supplied in the medium and the number of unlabelled bacterial cells added to start growth is kept to a minimum.

The advantages of expression in vivo

Labelling of QconCAT proteins in vivo is extremely robust, gives excellent yields and is more economical than using a cell-free labelling system in vitro.

References

1) Pratt, J. M., Simpson, D. M., Doherty, M. K., Rivers, J., Gaskell, S. J. and Beynon, R. J. (2006). Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes. Nature protocols, 1, No. 2, 1-15.

2) 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).

3) Rivers, J., Simpson, D. M., Robertson, D. H., Gaskell, S. J. and Beynon, R. J. (2007) Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT. Mol. Cell. Proteomics 6, 1416-1427.