Absolute quantification experiments usually use heavy-isotope labelled surrogate protein or peptide standards. For robust, multiplexed, high throughput experiments, QconCATs have the advantage of being simple to use and are less expensive than synthetic/AQUA peptides.

 

Quantitative proteomics requires differentially labelled analyte and standards. Standards may be provided as intact proteins, chemically synthesised peptides (AQUA peptides) or as concatamers of tryptic peptides in an artificial protein (QconCATs) (1,2). Each of these standards can be produced in heavy isotope-labelled form for absolute quantification experiments.

Intact proteins

The true internal standard for quantification of individual proteins in a proteomics study would ideally be the accurately quantified, corresponding protein, expressed in pure, isotope-labelled form, carrying the same post-translational modifications as the analyte. To generate such proteins would be very challenging, time consuming and expensive and is only feasible for very small numbers of proteins. Intact protein standards, lacking post-translational modifications, have however been used successfully in absolute quantification experiments where the aim was to quantify only very small numbers of proteins (3,4,5).

Chemically synthesised AQUA peptides

Most peptides can be chemically synthesised using well-established methods, however, there are reports of peptides that are refractory to chemical synthesis (6). Moreover, each chemically synthesised peptide must be purified and quantified separately and peptides have a tendency to adhere to surfaces, resulting in underestimations in quantification experiments. When quantifying large numbers of proteins the addition of each peptide comes with its own potential error. AQUA peptides, however, do not rely upon the efficiency of a tryptic digestion step and have been successfully employed in a number of studies (6,7).

QconCATs

QconCATs are artificial proteins that are concatamers of tryptic peptides (1,2,8). Purification and quantification of one QconCAT can result in as many as 100 peptides being released in a strict 1:1 stoichiometry after tryptic digestion. Operationally, therefore, it is much easier to purify, quantify and deliver many peptides in equimolar amounts to the analyte in one step. The performance of QconCATs relies upon the efficiency of the tryptic digestion, to ensure quantitative release of all the peptides. Special attention is always given to monitoring the digestion kinetics of QconCATs both alone and when mixed with the analyte (see The importance of complete proteolysis), however, due to their lack of higher order structure, QconCATs are normally readily digested to completion (8).

QconCATs versus AQUA peptides

Studies comparing the performance of AQUA and QconCAT in absolute quantification have shown the two approaches to be comparable (6,8). For the absolute quantification of a handful of proteins (around eight) it is probably more economical to use heavy isotope-labelled AQUA peptides, however for larger numbers of proteins QconCATs become increasingly more practical and economical. These calculations are however based on one peptide for each analyte, and the lack of an independent measure means that there can be no cross-validation. We would propose that for critical studies, a QRL (quantum replication level, the number of Q-peptides per analyte protein embedded in the QconCAT) should be at least 2. Under these circumstances, the cost advantage of QconCATs is clear.

References

1) Beynon, R.J. & Bond, J.S. (2001) Proteolytic enzymes, A Practical Approach. Oxford University Press, Oxford.

2) 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.

3) Peng, J., Kim, M. J., Cheng, D., Duong, D. M., Gygi, S. P. and Sheng, M. (2004) Semiquantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry. J. Biol. Chem., 279, 21003-11.

4) Brun, V., Dupuis, A., Adrait, A., Marcellin, M., Thomas, D., Court, M., Vandenesch, F. and Garin, J. (2007) Isotope-labeled protein standards: Towards absolute quantitative proteomics. Mol. Cell. Proteomics

5) Hanke, S., Besir, H., Oesterhelt, D. and Mann, M. (2008). Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level. J. Proteome Res., 7, 1118-1130.

6) Mirzaei, H., McBee, J., Watts, J. and Aebersold, R. (2007) Comparative evaluation of current peptide production platforms used in absolute quantification in proteomics. Mol. Cell. Proteomics Dec 2007; doi:10.1074/mcp.M700495-MCP200.

7) Barnidge, D. R., Dratz, E. A., Martin, T., Bonilla, L. E., Moran, L. B. and Lindall, A (2003). Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards. Ana., Chem.,75, 445-451.

8)  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.