Protein quantification that uses surrogate peptide standards, whether delivered as QconCAT proteins or as AQUA peptides, is critically dependent on complete proteolysis to release the analyte and for QconCAT the standard peptides.

Quantitative proteomics based on differentially labelled analyte and standards is predicated on the assumption that both the analyte and the standard are present in the analysis mixture in maximal amounts, which requires that the analyte (a sample that is a mixture of proteins) and the QconCAT are completely digested.

Monitoring digestion of the analyte

The incomplete digestion of the analyte is as significant an issue for AQUA peptides as for QconCATs and conditions of digestion should always be optimised (1-4). QconCATs can be used as tools to monitor the kinetics of release of the Q-peptide from the analyte and can therefore help in seeking optimal conditions for this process. QconCAT, digested to completion, can be added to analyte and samples taken for analysis by MS during incubation with trypsin (3,4). The signal intensity of the isotopically labelled Q-peptide from the QconCAT will remain constant, while that of the analyte derived unlabelled peptide will increase until a constant isotope ratio between the light (analyte) and heavy (QconCAT) Q-peptide is achieved, thus comparison between different conditions will identify those that are optimal.

Monitoring digestion of the QconCAT

Once the digestion conditions for the analyte have been optimised, the QconCAT should be digested on its own, using the same conditions. We have studied the cleavage kinetics of a number of QconCATs and have found that, in general, cleavage by trypsin is rapid and faster than the corresponding analyte (3,4). The QconCATs, being artificial proteins, are not predicted to form significant regions of secondary structure. Occasionally specific bonds within a QconCAT are cleaved more slowly than the corresponding analyte, but this is rare, and can be managed by optimal assembly of Q-peptides in a QconCAT to preserve the primary sequence context of each protein as much as is feasible (3,4). Since it is a simple experiment to confirm that the QconCAT is digested to completion using the conditions intended for analyte digestion, this step is considered as standard prior to proceeding to an absolute quantification experiment.

References

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

2) Hubbard, S.J., Beynon, R.J. & Thornton, J.M. (1998) Prediction of limited proteolytic sites in native protein structures from conformational parameters. Prot. Eng. 11, 349-359.

3) 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.

4) 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.