Absolute quantification approaches in proteomics automatically yield the stoichiometries of the proteins in the analyte. After prior isolation of subcellular particles, protein complexes or multimeric proteins, QconCATs can be used to quantify changes in stoichiometry in response to stimuli.

Protein composition of subcellular particles can change in response to a variety of stimuli. To be able to monitor such changes and to define the subunit stoichiometry of multiprotein complexes, absolute quantification approaches are required (1).

Identifying the proteins in subcellular particles

There are several methods that can be employed for the routine isolation of subcellular particles, or multiprotein complexes. These include employing differential centrifugation and/or immunoprecipitation using affinity tags. The protein compliment of these fractions is then readily identified using analysis by 1 or 2D SDS-PAGE, followed by in gel tryptic digestion and extraction of peptides (or by gel-free in-solution digestion) followed by LC-MS/MS. Repeat analyses after treatment of the starting tissue or cells with a variety of stimuli may identify that proteins change in type or abundance. The next step is to determine the stoichiometry of the proteins that are permanent or transient members of these complexes and how the proteins change in response to stimuli.

Designing and preparing a QconCAT protein

A simple example of how QconCATs can be used to define subunit stoichiometry of a protein complex is shown in Figure 1. Consider a protein complex comprised of four proteins A (15,000Da), B (25,000Da), C (20,000Da) and D (10,000Da) for which the stoichiometry can only be approximated from SDS-PAGE analysis. 1-2 tryptic peptides are selected as surrogate Q-peptides for each protein and a QconCAT protein comprising these peptides is designed, generated in heavy isotope-labelled form, fully characterised and quantified. For critical research, a Q-peptide Replication Level (see Designing a QconCAT) of greater than unity is preferred.

Determining stoichiometry using a QconCAT

For an absolute quantification experiment, a known amount of labelled QconCAT protein is mixed with a known amount of protein complex and tryptic digestion is carried out under optimised conditions. The digest is then analysed by e.g. LC-MS preferably using multiple reaction monitoring. Peak intensities for matched labelled (QconCAT) and unlabelled (protein complex) peptides are then recorded and the absolute amount of each protein under investigation is calculated in nmol/g protein complex. This reveals the stoichiometry of the proteins in the original protein complex (2,3).

 

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) Kito, K., Ota, K., Fujita, T. and Ito, T. (2007) A Synthetic Approach toward Accurate mass Spectrometric Quantification of Component Stoichiometry of Multiprotein Complexes. J. Proteome Res. 6, 792-800.

3) Nanavati, D., Gucek, M., Milne, J. L. S., Subramanian, S. and Markey, S. P., (2007). Stoichiometry and absolute quantification of proteins with mass spectrometry using fluorescent and isotope labelled concatenated peptide standards. Mol. Cell. Proteomics 7, 442 – 447.